Cell composition and methods of making the same

ABSTRACT

Provided herein are improved methods for the formulation of compositions comprising placental stem cells, and improved compositions and cell formulations produced thereby.

This application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/090,577, filed Aug. 20, 2008, which ishereby incorporated by reference in its entirety.

1. FIELD

Provided herein are improved compositions, e.g., pharmaceuticalcompositions, comprising cells, e.g., stem cells and placental cells,such as isolated human adherent placental multipotent cells, e.g., theplacental multipotent cells described in section 5.3, or cells isolatedfrom placental perfusate, e.g., total nucleated cells isolated fromplacental perfusate, and improved methods for making the compositions.

2. BACKGROUND

Cell compositions, e.g., stem cell compositions, have become anattractive therapy for a number of physiological deficiencies, e.g.,bone marrow replacement. A need exists for improved formulations ofcells, e.g., stem cells, that are to be administered to individuals inneed of such compositions.

3. SUMMARY

Provided herein are improved methods of making compositions comprisingcells, e.g., isolated placental cells, such as placental stem cells,placental multipotent cells, placental cells that can be expanded andhave the potential to differentiate into at least two different celltypes, e.g., osteogenic and chondrogenic cell types, or cells isolatedfrom placental perfusate, e.g., total nucleated cells isolated fromplacental perfusate, and compositions comprising such cells, e.g., thatare suitable for administration to an individual. The improved methodsuse specific steps and specific compositions for thepre-cryopreservation treatment, cryopreservation, and thawing of cells.In certain embodiments, the improved methods reduce or eliminatepost-thaw clumping of cryopreserved cells. In preferred embodiments, theimproved compositions comprise placental multipotent cells.

In one embodiment, provided herein is a method of making a compositioncomprising: (a) contacting cells with a solution comprising dextran andhuman serum albumin (HSA) to form a cell-containing solution; (b)filtering the cell-containing solution to form a filteredcell-containing solution; (c) optionally diluting the filteredcell-containing solution to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶,1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter with afirst dilution solution comprising dextran; and (d) optionally dilutingthe filtered cell-containing solution with a second dilution solutioncomprising dextran. In some embodiments, step (c) is performed where thefiltered cell-containing solution in (b) comprises greater than about15×10⁶ cells per milliliter, wherein said diluting in step (c) is toabout 15×10⁶ cells per milliliter. In particular embodiments, step (c)is performed where the filtered cell-containing solution in (b)comprises greater than about 10±3×10⁶ cells per milliliter, wherein saiddiluting in step (c) is to about 10±3×10⁶ cells per milliliter. In someembodiments, step (c) is performed where the filtered cell-containingsolution in (b) comprises greater than about 7.5×10⁶ cells permilliliter, wherein said diluting in step (c) is to about 7.5×10⁶ cellsper milliliter. In a specific embodiment, the solution comprisingdextran of step (d) does not comprise human serum albumin. In a specificembodiment, the cells of the filtered cell-containing composition arecryopreserved prior to step (d). In certain embodiments, if the numberof cells is less than about 10±3×10⁶ cells per milliliter following step(a), filtration is optional. In certain embodiments, if the number ofcells is less than about 7.5×10⁶ cells per milliliter following step(a), filtration is optional.

In some embodiments, said dextran in said first dilution solution orsaid second solution is 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%,7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%,9.75%, or 10% dextran. In some embodiments, said dextran in said firstdilution solution or said second solution is about 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19% or 20% dextran. The dextran in the firstdilution solution or second dilution solution can be dextran of anymolecular weight, e.g., dextran having a molecular weight of from about1 kDa to about 150 kDa, about 1 kDa to about 125 kDa, about 1 kDa toabout 100 kDa, about 1 kDa to about 75 kDa, about 1 kDa to about 50 kDa,or about 1 kDa to about 25 kDa. In some embodiments, the dextran in thefirst dilution solution or second dilution solution has a molecularweight of about 1 kDA to about 10 kDa, about 30 kDa to about 50 kDa, orabout 60 kDa to about 80 kDa. In another specific embodiment, saiddextran in said first dilution solution or said second dilution solutionis dextran 1. In another specific embodiment, said dextran in said firstdilution solution and said second dilution solution is dextran 1. Inanother specific embodiment, said dextran in said first dilutionsolution or said second dilution solution is dextran 70. In anotherspecific embodiment, said dextran in said first dilution solution andsaid second dilution solution is dextran 70. In another specificembodiment, said dextran in said first dilution solution or said seconddilution solution is dextran 40. In another specific embodiment, saiddextran in said first dilution solution and said second dilutionsolution is dextran 40. In another specific embodiment, said dextran 40in said first dilution or said second dilution solution is 2.5% to 10%dextran 40. In some embodiments, said dextran 40 in said first dilutionsolution or said second solution is about 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10% dextran 40. In some embodiments, saiddextran in said first dilution solution or said second solution is about11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% dextran 40. Inanother specific embodiment, said dextran 40 in said first dilutionsolution is 5.0% dextran 40. In another specific embodiment, saiddextran 40 in said first dilution solution is 5.5% dextran 40. Inanother specific embodiment, said dextran 40 in said second dilutionsolution is 10% dextran 40.

In other embodiments, said first and/or second dilution solutions maycomprise a polysaccharide in addition to or other than, i.e., in placeof, dextran. For example, in some embodiments, said first and/or seconddilution solutions comprises maltodextrin (e.g., about 2.5%, 2.75%,3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%,5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%,8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% maltodextrin), trehalose(e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%,4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%,7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10%trehalose), or hetastarch (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% hetastarch). In other embodiments, said firstand/or second dilution solutions comprises sucrose (e.g., about 2.5%,2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%,5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%,8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% sucrose), heparin(e.g., 55 USP units/mL heparin), or glycogen (e.g., about 2.5%, 2.75%,3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%,5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%,8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% glycogen). In a particularembodiment, said first and/or second dilution solutions comprisesmaltodextran in addition to or other than, i.e., in place of, dextran.In another particular embodiment, said first and/or second dilutionsolutions comprises trehalose in addition to or other than, i.e., inplace of, dextran. In another particular embodiment, said first and/orsecond dilution solutions comprises hetastarch in addition to or otherthan, i.e., in place of, dextran.

In another specific embodiment, said HSA in said solution comprising HSAis about 1 to 17% HSA. In another specific embodiment, said HSA in saidsolution comprising HSA is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16% or 17% HSA. In another specificembodiment, said HSA in said solution comprising HSA is about 4 to 10%HSA. In another specific embodiment, said HSA in said solutioncomprising HSA is about 3.125% HSA. In another specific embodiment, saidHSA in said solution comprising HSA is about 5% HSA. In another specificembodiment, said HSA in said solution comprising HSA is about 10% HSA.In another specific embodiment, said HSA in said solution comprising HSAis about 16.875% HSA. In another specific embodiment, said HSA in saidfirst dilution solution is about 1 to 17% HSA. In another specificembodiment, said HSA in said first dilution solution is about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% or 17%HSA. In another specific embodiment, said HSA in said first dilutionsolution is about 4 to 10% HSA. In another specific embodiment, said HSAin said first dilution solution is about 3.125% HSA. In another specificembodiment, said HSA in said first dilution solution is about 5% HSA. Inanother specific embodiment, said HSA in said first dilution solution isabout 10% HSA. In another specific embodiment, said HSA in said firstdilution is about 16.875% HSA.

In other embodiments, bovine serum albumin (BSA)(e.g., about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% BSA) or fetalbovine serum (FBS) (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14% or 15% FBS) may be used in addition to or in placeof, i.e., instead of HSA in said solution.

In some embodiments, the ratio of HSA to dextran, e.g., dextran 1,dextran 40 or dextran 70, in the first solution is between about 6:1HSA:dextran to about 1:2.6 HSA:dextran. In some embodiments, the ratioof HSA to dextran is about 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1,2.5:1, 2.0:1, 1.5:1, 1:1, 1:1.5, 1:2 or 1:2.6 HSA:dextran. In someembodiments, the ratio of HSA to dextran, e.g., dextran 1, dextran 40 ordextran 70, in the first solution is about 3.13% HSA/8.25% dextran. Insome embodiments, the ratio of HSA to dextran, e.g., dextran 1, dextran40 or dextran 70, in the first solution is about 16.88% HSA/2.75%dextran. In particular embodiments, the ratio of HSA to dextran, e.g.,dextran 1, dextran 40 or dextran 70, in the first solution is about 10%HSA/5.5% dextran, e.g., dextran 1, dextran 40 or dextran 70.

In another specific embodiment, said solution in step (a) orcell-containing solution comprises a cryoprotectant. In a more specificembodiment, said cryoprotectant is dimethylsulfoxide (DMSO). In aparticular embodiment, the solution recited in step (a) comprises about1% to about 15%, about 2.5% to about 15%, about 2.5% to about 10%, about5% to about 15%, about 5% to about 10% or about 10% to about 15% DMSO.In a particular embodiment, the solution recited in step (a) comprisesabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%DMSO. In a particular embodiment, the solution recited in step (a)comprises about 5% DMSO. In another specific embodiment, said firstdilution solution further comprises a cryoprotectant. In a more specificembodiment, said cryoprotectant is dimethylsulfoxide (DMSO). In aparticular embodiment, said first dilution solution further comprisesabout 1% to about 15%, about 2.5% to about 15%, about 2.5% to about 10%,about 5% to about 15%, about 5% to about 10% or about 10% to about 15%DMSO. In a particular embodiment, said first dilution solution furthercomprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14% or 15% DMSO. In a particular embodiment, said first dilutionsolution further comprises about 5% DMSO.

In a specific embodiment, said first dilution solution comprises about5.5% dextran 40, about 10% HSA, and about 5% DMSO.

In a particular embodiment, said method produces a compositioncomprising cells and about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%,4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%,7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%,9.75%, or 10% dextran, e.g., dextran 1, dextran 40 or dextran 70. Inanother particular embodiment, said method produces a compositioncomprising cells and about 7.5% to about 9% dextran, e.g., dextran 40.In another specific embodiment, said method produces a compositioncomprising about 1.5×10⁶ cells per milliliter to about 3.75×10⁶ cellsper milliliter. In another specific embodiment, said method produces acomposition comprising about 1.0±0.3×10⁶ cells per milliliter to about5.0±1.5×10⁶ cells per milliliter. In other specific embodiments, themethod produces a composition comprising between about 1.0×10⁶ cells permilliliter and 15×10⁶ cells per milliliter, e.g., between about 7.5×10⁶cells per milliliter and about 15×10⁶ cells per milliliter. In anotherspecific embodiment, said method produces a composition comprising fromabout 1% HSA to about 15% HSA. In another specific embodiment, saidmethod produces a composition comprising from about 1% HSA to about 10%HSA.

Further provided herein is a method of making a composition, comprising:(a) filtering a plurality of cells in a solution comprising 5.5% dextran40 and 10% HSA through a 70 μM-150 μM filter to form a filteredcell-containing solution; (b) optionally diluting the filteredcell-containing solution with 5.5% dextran 40, 10% HSA, and 5% DMSO toabout 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to 15×10⁶,or 1 to 10×10⁶ cells per milliliter; (c) cryopreserving the cells; (d)thawing the cells; and (e) diluting the filtered cell-containingsolution with 10% dextran 40 to produce said composition. In someembodiments, step (b) is performed where the filtered cell-containingsolution in (a) comprises greater than about 15×10⁶ cells permilliliter, wherein said diluting in step (b) is to about 15×10⁶ cellsper milliliter. In certain embodiments, if the filtered cell-containingsolution in (a) comprises less than about 15×10⁶ cells per milliliter,filtration is optional. In some embodiments, step (b) is performed wherethe filtered cell-containing solution in (a) comprises greater thanabout 10+3×10⁶ cells per milliliter, wherein said diluting in step (b)is to about 10±3×10⁶ cells per milliliter. In certain embodiments, ifthe filtered cell-containing solution in (a) comprises less than about10±3×10⁶ cells per milliliter, filtration is optional. In someembodiments, step (b) is performed where the filtered cell-containingsolution in (a) comprises greater than about 7.5×10⁶ cells permilliliter, wherein said diluting in step (b) is to about 7.5×10⁶ cellsper milliliter. In certain embodiments, if the filtered cell-containingsolution in (a) comprises less than about 7.5×10⁶ cells per milliliter,filtration is optional. In some embodiments, step (e) comprises dilutingthe filtered cell-containing solution 1:1 to 1:5 (v/v) with 10% dextran40. In some embodiments, step (e) comprises diluting the filteredcell-containing solution 1:1 to 1:11 (v/v) with 10% dextran 40. In amore specific embodiment, the cell-containing solution of step (a)additionally comprises a cryoprotectant, e.g., DMSO, e.g., about 2% toabout 15% DMSO. In a particular embodiment, the solution recited in step(a) additionally comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14% or 15% DMSO. In a particular embodiment, thesolution recited in step (a) additionally comprises about 5% DMSO. In apreferred embodiment, the filter in step (a) is a 70 μM to 100 μMfilter.

In another embodiment, provided herein is a method of making acomposition, comprising: (a) centrifuging a plurality of cells tocollect the cells; (b) resuspending the cells in 5.5% dextran 40; (c)centrifuging the cells to collect the cells; (d) resuspending the cellsin a 5.5% dextran 40 solution that comprises 10% HSA to produce acell-containing solution; (e) filtering the cell-containing solutionthrough a 40 μM to 150 μM filter to produce a filtered cell-containingsolution; (f) optionally diluting the filtered cell-containing solutionin 5.5% dextran 40, 10% HSA, and a cryoprotectant, e.g., DMSO, e.g., 5%DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to15×10⁶, or 1 to 10×10⁶ cells per milliliter; (g) cryopreserving thecells; (h) thawing the cells; and (i) diluting the cell-containingsolution 1:1 to 1:11 (v/v) with 10% dextran 40 to produce saidcomposition. In some embodiments, step (f) is performed where thefiltered cell-containing solution in (e) comprises greater than about15×10⁶ cells per milliliter, wherein said diluting in step (f) is toabout 15×10⁶ cells per milliliter. In particular embodiments, step (f)is performed where the filtered cell-containing solution in (e)comprises greater than about 10±3×10⁶ cells per milliliter, wherein saiddiluting in step (f) is to about 10±3×10⁶ cells per milliliter. In someembodiments, step (e) is performed where the filtered cell-containingsolution in (e) comprises greater than about 7.5×10⁶ cells permilliliter, wherein said diluting in step (0 is to about 7.5×10⁶ cellsper milliliter. In certain embodiments, if the number of cells is lessthan about 10±3×10⁶ cells per milliliter following step (d), filtrationis optional. In certain embodiments, if the number of cells is less thanabout 7.5×10⁶ cells per milliliter following step (d), filtration isoptional. In a particular embodiment, if the recited resuspending instep (d) would result in a cell-containing solution comprising less thanabout 10±3×10⁶ cells per milliliter, the solution recited in step (d)comprises a cryoprotectant, e.g., DMSO, e.g., about 2% to about 15%DMSO, and step (f) is not performed. In a preferred embodiment, thefilter in step (e) is a 70 μM to 100 μM filter.

Also provided herein is a method of making a composition, comprising:(a) filtering a solution comprising isolated placental cells, 5.5%dextran 40 and 10% human serum albumin (HSA) with a filter that removesvisible cell clumps to produce a filtered isolated placentalcell-containing solution; (b) optionally diluting said filtered isolatedplacental cell-containing solution with an amount of a solutioncomprising 5.5% dextran 40, 10% HSA and 5% dimethylsulfoxide (DMSO)sufficient to bring said filtered isolated placental cell-containingsolution to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1to 15×10⁶, or 1 to 10×10⁶ cells per milliliter; and (c) diluting saidfiltered isolated placental cell-containing solution with 10% dextran 40to produce said composition. In some embodiments, step (b) is performedwhere the filtered cell-containing solution in (a) comprises greaterthan about 15×10⁶ cells per milliliter, wherein said diluting in step(b) is to about 15×10⁶ cells per milliliter. In particular embodiments,step (b) is performed where the filtered cell-containing solution in (a)comprises greater than about 10±3×10⁶ cells per milliliter, wherein saiddiluting in step (b) is to about 10±3×10⁶ cells per milliliter. In someembodiments, step (b) is performed where the filtered cell-containingsolution in (a) comprises greater than about 7.5×10⁶ cells permilliliter, wherein said diluting in step (b) is to about 7.5×10⁶ cellsper milliliter. In some embodiments, step (c) comprises diluting saidfiltered isolated placental cell-containing solution with 10% dextran 40at a ratio of about 1:1 to about 1:11 isolated placental cell-containingsolution:dextran 40 (v/v). In some embodiments, step (c) comprisesdiluting said filtered isolated placental cell-containing solution with10% dextran 40 at a ratio of about 1:1 to about 1:5 isolated placentalcell-containing solution:dextran 40 (v/v). In certain embodiments, ifthe number of cells is less than about 10±3×10⁶ cells per milliliter,filtration is optional. In certain embodiments, if the number of cellsfollowing step (a) is less than about 7.5×10⁶ cells per milliliter,filtration is optional. In a specific embodiment, said filter is a 70 μMfilter. In another specific embodiment, said filter is a 100 μM filter.In another specific embodiment, the filter in step (a) is a 70 μM to 100μM filter.

In a specific embodiment of any of the above methods, the composition isa pharmaceutical composition.

In another specific embodiment of any of the above methods, the methodfurther comprises concentrating the resulting cell composition to about5×10⁶ cells per milliliter to 1×10⁸ cells per milliliter. Such acomposition is useful, for example, for subcutaneous administration ofthe composition to an individual in need thereof.

In another aspect, provided herein are compositions, e.g.,pharmaceutical compositions comprising cells, e.g., stem cells, isolatedplacental cells, e.g., placental stem cells or placental multipotentcells. In certain embodiments, the compositions are made by any of themethods described herein. In one embodiment, provided herein is acomposition, e.g., a solution, comprising a plurality of cells, e.g.,stem cells, isolated placental cells, for example, placental stem cellsor placental multipotent cells, wherein said composition comprisesbetween about 1.0±0.3×10⁶ cells per milliliter to about 5.0+1.5×10⁶cells per milliliter, and wherein said composition comprises no visiblecell clumps (i.e., no macro cell clumps), or substantially no suchvisible clumps. In certain other embodiments, the composition comprisesbetween about 1.0×10⁶ cells per milliliter and 15×10⁶ cells permilliliter, e.g., between about 7.5×10⁶ cells per milliliter and about15×10⁶ cells per milliliter. In certain other embodiments, thecomposition comprises less than about 20×10⁶ cells per milliliter.

In some embodiments, said composition comprises about 2.5%, 2.75%, 3.0%,3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%,6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%,8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% dextran, e.g., dextran 1,dextran 40 or dextran 70. In a specific embodiment, said compositioncomprises about 7.5% to about 9% dextran 40. In a specific embodiment,said composition comprises about 5.5% dextran 40.

In other embodiments, said composition comprises a polysaccharide inaddition to or other than, i.e., in place of, dextran. In certainembodiments, the polysaccharide is a polymer of glucose that does notcomprise non-glucose saccharide subunits. In other embodiments, saidcomposition comprises maltodextrin (e.g., about 2.5%, 2.75%, 3.0%,3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%,6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%,8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% maltodextrin), trehalose (e.g.,about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%,5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%,7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10%trehalose), or hetastarch (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% hetastarch). In other embodiments, saidcomposition comprises sucrose (e.g., about 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10% sucrose), heparin (e.g., 55 USP/mlheparin), or glycogen (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% glycogen). In a particular embodiment, saidcomposition comprises maltodextran in addition to or other than, i.e.,in place of, dextran. In another particular embodiment, said compositioncomprises trehalose in addition to or instead of dextran. In anotherparticular embodiment, said composition comprises hetastarch in additionto or instead of dextran.

In another specific embodiment, said composition comprises about 1% toabout 17% HSA. In some embodiments, said composition comprises about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, orabout 17% HSA. In some embodiments, said composition comprises about3.125% HSA. In some embodiments, said composition comprises about 5%HSA. In some embodiments, said composition comprises about 10% HSA. Insome embodiments, said composition comprises about 16.875% HSA.

In other embodiments, said composition comprises bovine serum albumin(BSA)(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14% or 15% BSA) or fetal bovine serum (FBS) (e.g., about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% FBS) inaddition to or instead of HSA.

In some embodiments, said composition comprises a cryoprotectant, e.g.,DMSO, e.g., about 1% to about 15% DMSO. In some embodiments, saidcomposition comprises about 1% to about 5% DMSO. In some embodiments,said composition comprises about 1% to about 15%, about 2.5% to about15%, about 2.5% to about 10%, about 5% to about 15%, about 5% to about10% or about 10% to about 15% DMSO. In some embodiments, saidcomposition comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14% or 15% DMSO. In a particular embodiment, thecomposition comprises about 5% DMSO.

In a specific embodiment, said cells have been cryopreserved and thawed.In another specific embodiment, said cells have been filtered through a70 μM to 100 μM filter. In another specific embodiment, said compositioncomprises no visible cell clumps. In another specific embodiment, saidcomposition comprises fewer than about 200 cell clumps per 10⁶ cells,wherein said cell clumps are visible only under a microscope, e.g., alight microscope. In another specific embodiment, said compositioncomprises fewer than about 150 cell clumps per 10⁶ cells, wherein saidcell clumps are visible only under a microscope, e.g., a lightmicroscope. In another specific embodiment, said composition comprisesfewer than about 100 cell clumps per 10⁶ cells, wherein said cell clumpsare visible only under a microscope, e.g., a light microscope.

In a specific embodiment of any of the methods or compositions describedherein, the cells are stem cells, for example, stem cells isolated froma human postpartum placenta that has been drained of blood. In certainembodiments, such cells have been expanded. In another specificembodiment of any of the above embodiments, the cells are adherentcells, that is, cells that adhere to a tissue culture surface, e.g.,tissue culture plastic (either uncoated or coated with, e.g.,fibronectin, laminin, or the like). Examples of adherent cells include,e.g., adherent placental stem cells, as described herein; bonemarrow-derived mesenchymal stem cells, fibroblasts, or the like. Inanother embodiment, the cells are human cells.

In another embodiment, said cells are cells obtained from (e.g.,isolated from) placental perfusate. In a more specific embodiment, saidcells are nucleated cells, e.g., total nucleated cells, obtained fromplacental perfusate. In certain embodiments, the placenta from whichtotal nucleated placental cells are obtained by perfusion is drained ofblood and perfused to remove residual blood prior to perfusion tocollect total nucleated placental cells. In certain other embodiments,the placenta from which total nucleated placental cells are obtained byperfusion is drained of blood but is not perfused to remove residualblood prior to perfusion to collect total nucleated placental cells. Incertain other embodiments, the placenta from which total nucleatedplacental cells are obtained by perfusion is neither drained of bloodnor perfused to remove residual blood prior to perfusion to collecttotal nucleated placental cells.

In another specific embodiment of the method, said cells are stem cells.In more specific embodiments, the stem cells are adult stem cells,somatic stem cells, embryonic stem cells, embryonic germ cells,umbilical cord stem cells, amniotic fluid stem cells, bonemarrow-derived mesenchymal stem cells, cord blood-derived mesenchymalstem cells, peripheral blood-derived mesenchymal stem cells,adipose-derived mesenchymal stem cells or periosteum-derived mesenchymalstem cells. In another embodiment, said cells are natural killer cells.

In another specific embodiment, said cells are isolated placental cells.In certain embodiments, the isolated placental cells are isolatedplacental stem cells. In certain other embodiments, the isolatedplacental cells are isolated placental multipotent cells.

In certain embodiments, the isolated placental cells are isolatedplacental stem cells. In certain other embodiments, the isolatedplacental cells are isolated placental multipotent cells. In a specificembodiment, the isolated placental cells are CD34⁻, CD10⁺ and CD105⁺ asdetected by flow cytometry. In a more specific embodiment, the isolatedCD34⁻, CD10⁺, CD105⁺ placental cells are placental stem cells. Inanother more specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺placental cells are multipotent placental cells. In another specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells have thepotential to differentiate into cells of a neural phenotype, cells of anosteogenic phenotype, or cells of a chondrogenic phenotype. In a morespecific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cellsare additionally CD200⁺. In another more specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ placental cells are additionally CD90⁺ orCD45⁻, as detected by flow cytometry. In another more specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells areadditionally CD90⁺ or CD45⁻, as detected by flow cytometry. In a morespecific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cellsare additionally CD90⁺ or CD45⁻, as detected by flow cytometry. Inanother more specific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ cellsare additionally CD90⁺ and CD45⁻, as detected by flow cytometry. Inanother more specific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺,CD90⁺, CD45⁻ cells are additionally CD80⁻ and CD86⁻, as detected by flowcytometry.

In a more specific embodiment, the CD34⁻, CD10⁺, CD105⁺ cells areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, CD80⁻, CD86⁻,SH3⁺ or SH4⁺. In another more specific embodiment, the cells areadditionally CD44⁺. In a specific embodiment of any of the isolatedCD34⁻, CD10⁺, CD105⁺ placental cells above, the cells are additionallyone or more of CD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺,HLA-DP,DQ,DR⁻, and/or Programmed Death-1 Ligand (PDL1)⁺.

In other embodiments, the isolated placental cells are CD200⁺ andHLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ andHLA-G⁺; CD73⁺ and CD105⁺ and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells comprising saidisolated placental cells when said population is cultured underconditions that allow the formation of an embryoid-like body; or OCT-4⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising the isolated placental cellswhen said population is cultured under conditions that allow formationof embryoid-like bodies; or any combination thereof. In a specificembodiment, said CD200⁺, HLA-G⁺ placental cells are CD34⁻, CD38⁻, CD45⁻,CD73⁺ and CD105⁺. In another specific embodiment, said CD73⁺, CD105⁺,and CD200⁺ placental cells are CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. Inanother specific embodiment, said CD200⁺, OCT-4⁺ placental cells areCD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In another specificembodiment, said CD73⁺, CD105⁺ and HLA-G⁺ placental cells are CD34⁻,CD45⁻, OCT-4⁺ and CD200⁺. In another specific embodiment, said CD73⁺ andCD105⁺ placental cells are OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said placental cells are CD73⁺, CD105⁺, CD200⁺,CD34⁻, CD38⁻, and CD45⁻.

In certain embodiments, the isolated placental cells are one or more ofCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD80⁻, CD86⁻, CD90⁺,CD117⁻, CD133⁻, CD200⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺,MHC-I⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, PDL1⁺ or ABC-p⁺,where ABC-p is a placenta-specific ABC transporter protein (also knownas breast cancer resistance protein (BCRP) and as mitoxantroneresistance protein (MXR)). In a specific embodiment, the isolatedplacental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺,CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3-, SSEA4⁻, and OCT-4⁺. In anotherembodiment, the isolated placental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻,CD45⁻, CD54⁺, SH2⁺, SH3⁺, and SH4⁺. In another embodiment, the isolatedplacental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺,SH3⁺, SH4⁺ and OCT-4⁺. In another embodiment, the isolated placentalcells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺,HLA-1⁺, SH2⁺, SH3⁺, SH4⁺. In another embodiment, the isolated placentalcells are OCT-4⁺ and ABC-p⁺. In another embodiment, the isolatedplacental cells are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. In another embodiment,the isolated placental cells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻. In aspecific embodiment, said OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻ cells areadditionally CD10⁺, CD29⁺, CD34+, CD44⁻, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, and SH4⁺. In another embodiment, the isolated placental cells areOCT-4⁺ and CD34⁻, and either SH3⁺ or SH4⁺. In another embodiment, theisolated placental cells are CD34⁻ and either CD10⁺, CD29⁺, CD44⁺,CD54⁺, CD90⁺, or OCT-4⁺. In certain embodiments, the isolated placentalcells are CD10⁺, CD34+, CD105⁺ and CD200⁺.

In another embodiment, the isolated placental cells useful in themethods of treatment described herein are one or more of CD10⁺, CD29⁻,CD44⁺, CD45⁻, CD54/ICAM⁻, CD62-E⁻, CD62-L⁻, CD62-P⁻, CD80⁻, CD86⁻,CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(low),CD184/CXCR4⁻, β2-microglobulin^(low), MHC-I^(low), MHC-II⁻, HLA-G^(low),and/or PDL1^(low). In a specific embodiment, the isolated placentalcells are at least CD29⁻ and CD54⁻. In another specific embodiment, theisolated placental cells are at least CD44⁺ and CD106⁺. In anotherspecific embodiment, the isolated placental cells are at least CD29⁺.

In another specific embodiment, said isolated placental cells expressone or more genes at a detectably higher level than an equivalent numberof bone marrow-derived mesenchymal stem cells, wherein said one or moregenes are one or more of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE,C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1,FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18,KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3,PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN,and ZC3H12A, and wherein said bone marrow-derived mesenchymal stem cellshave undergone a number of passages in culture equivalent to the numberof passages said isolated placental cells have undergone. In a morespecific embodiment, said isolated placental cells express said one ormore genes when cultured for about 3 to about 35 population doublings ina medium comprising 60% Dulbecco's Modified Eagle's Medium (DMEM)-LG(preferably from Gibco) and 40% MCDB-201 (preferably from Sigma); 2%fetal calf serum (preferably from Hyclone Labs); 1×insulin-transferrin-selenium (ITS); 1× linoleic acid-bovine serumalbumin (LA-BSA); 10⁻⁹ M dexamethasone (preferably from Sigma); 10⁻⁴ Mascorbic acid 2-phosphate (preferably from Sigma); epidermal growthfactor 10 ng/mL (preferably from R&D Systems); and platelet-derivedgrowth factor (PDGF-BB) 10 ng/mL (preferably from R&D Systems). In amore specific embodiment, said isolated placental cells express said oneor more genes when cultured for from about 3 to about 35 populationdoublings in a medium comprising 60% DMEM-LG (preferably from Gibco) and40% MCDB-201 (preferably from Sigma); 2% fetal calf serum (preferablyfrom Hyclone Labs); 1× insulin-transferrin-selenium (ITS); 1× linoleicacid-bovine serum albumin (LA-BSA); 10⁻⁹ M dexamethasone (preferablyfrom Sigma); 10⁻⁴ M ascorbic acid 2-phosphate (preferably from Sigma);epidermal growth factor 10 ng/mL (preferably from R&D Systems); andplatelet-derived growth factor (PDGF-BB) 10 ng/mL (preferably from R&DSystems).

In another specific embodiment, said placental stem cells express theneurotrophic growth factors glial cell derived neurotrophic factor(GDNF), brain-derived neurotrophic factor (BDNF), hepatocyte growthfactor (HGF), placental growth factor (PGF) and vascular endothelialgrowth factor (VEGF).

In another specific embodiment, said isolated placental cells arecontained within a population of cells, at least 50% of the cells ofwhich are said isolated placental cells. In another specific embodiment,said isolated placental cells are contained within a population ofcells, at least 70% of the cells of which are said isolated placentalcells. In another specific embodiment, said isolated placental cells arecontained within a population of cells, at least 80% of the cells ofwhich are said isolated placental cells. In another specific embodiment,said isolated placental cells are contained within a population ofcells, at least 90% of the cells of which are said isolated placentalcells. In certain other embodiments, the placental cells in saidpopulation of cells are substantially free of cells having a maternalgenotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% of the placental cells in said population havea fetal genotype, i.e., are fetal in origin. In certain otherembodiments, the population of cells comprising said placental cells aresubstantially free of cells having a maternal genotype; e.g., at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%of the cells in said population have a fetal genotype, i.e., are fetalin origin.

In certain embodiments, the isolated placental cells are CD34⁺ placentalcells, e.g., hematopoietic placental cells. Such cells are obtainablefrom placental tissue, e.g., from a placenta that has been drained ofcord blood and perfused to remove residual blood. In certainembodiments, the CD34⁺ placental cells are CD38⁺. In certainembodiments, the CD34⁺ placental cells are CD38⁻. In certain otherembodiments, the CD34⁺ placental cells are CD45⁺. In a specificembodiment, the placental cells are CD34⁺, CD38⁻ and CD45⁺.

In any of the above embodiments of isolated placental cells, theisolated placental cells generally do not differentiate during culturingin growth medium, i.e., medium formulated to promote proliferation,e.g., during proliferation in growth medium. In another specificembodiment, said isolated placental cells do not require a feeder layerin order to proliferate. In another specific embodiment, said isolatedplacental cells do not differentiate in culture as the result of culturein the absence of a feeder cell layer.

In another more specific embodiment, said isolated placental cells areobtained by perfusion of a post-partum placenta that has been drained ofblood and perfused to remove residual blood; drained of blood but notperfused to remove residual blood; or neither drained of blood norperfused to remove residual blood. In another more specific embodiment,said isolated placental cells are obtained by physical and/or enzymaticdisruption of placental tissue.

In certain embodiments of the above method, isolated placental cells arefiltered and cryopreserved as part of the construction of a placentalcell bank. For example, isolated placental cells are isolated from aplacenta, or placental tissue, and, after culturing, are resuspended ina solution comprising, e.g., dextran, e.g., dextran 40, e.g., 5.5%dextran 40. In more specific embodiments, the solution additionallycomprises HSA and/or DMSO, in preparation for cryopreservation.Cryopreserved isolated placental cells in the bank are, as needed,thawed and diluted with, e.g., a solution comprising 10% dextran 40 asdescribed herein. In certain embodiments of the method, the filtrationand dilution method described herein is not a part of the initialisolation of isolated placental cells.

In certain embodiments, said isolated placental cells are obtained byperfusion of a post-partum placenta that has been drained of blood andperfused to remove residual blood. In another more specific embodiment,said isolated placental cells are obtained by physical and/or enzymaticdisruption of placental tissue.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a ternary diagram depicting solution space of HSA,Dextran 40 and DMSO and experimental design for assessing the effect ofvarying component concentration on cell viability and proliferation.

FIG. 2 presents Filter Retention Assay (FRA) data for formulationscomprising different percentages of DMSO. Data are expressed in pixelsper million cells loaded (px/MM) as read on a Vi-Cell cell viabilityanalyzer. Assay control=100% dextran 40 solution, with cell stain,without cells.

FIG. 3 presents FRA data for cell formulations comprising differentvolume fractions of HSA. Data are expressed in pixels per million cellsloaded (px/MM) as read on a Vi-Cell cell viability analyzer. Assaycontrol=100% dextran 40 solution, with cell stain, without cells.

FIG. 4 presents post thaw trypan blue viability for cell formulationscomprising different percentages of DMSO (0-20%).

FIG. 5 presents post thaw total cell recovery as a function of varyingconcentrations of DMSO (0-20%).

FIG. 6: Culture re-establishment as a function of varying formulationscomprising different percentages of DMSO, as assessed by the MTS assay(see Section 6.3.1, below).

FIG. 7 presents post thaw cell viability of cell formulations comprisingdifferent volume fractions of 25% HSA.

FIG. 8 presents post thaw total cell recovery as a function of HSAvolume fraction.

FIG. 9 presents data assessing culture re-establishment as a function ofdifferent fractions of HSA, generated through use of a Cell Titer 96®Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison,Wis.).

FIG. 10: Levels of immunosuppression as assessed by a Bead ReactionAssay for formulations comprising different concentrations offormulation components.

FIG. 11: Cellular aggregation as a function of varying freezing celldensities (1−40×10⁶ cells/mL), as determined by FRA assay. Data areexpressed in pixels per million cells loaded (px/MM) as read on aVi-Cell cell viability analyzer.

FIG. 12 Cellular aggregation as a function of freezing cell densities(1−40 million cells/mL). Data are expressed in pixels per million cellsloaded (px/MM) as read on a Vi-Cell cell viability analyzer.

FIG. 13: Cellular aggregation as a function of varying molecular weightsof dextran, as determined by FRA assay. FRA signal equivalent acrossdextran 1,000, 40,000 and 70,000 (i.e., dextran 1, dextran 40 anddextran 70, respectively). Data are expressed in pixels per millioncells loaded (px/MM). Assay control=100% dextran 40 solution, with cellstain, without cells.

FIG. 14 presents post thaw viability across formulations comprisingdifferent molecular weights of dextran.

FIG. 15 presents cell recovery across formulations comprising differentmolecular weights of dextran.

FIG. 16 presents CD105⁺/CD200⁺ across formulations comprising differentmolecular weights of dextran.

FIG. 17 presents bead T cell reaction (BTR) data across formulationscomprising different molecular weights of dextran.

FIG. 18 presents cellular aggregation measured by FRA acrossformulations comprising different polysaccharides. Assay control=100%dextran 40 solution, with cell stain, without cells.

FIG. 19 presents post-thaw viability of cells formulated withnon-dextran 40 polysaccharides. Data are expressed in pixels per millioncells loaded (px/MM).

FIG. 20: Viable cell recovery as a function of formulations comprisingdifferent polysaccharides.

FIG. 21 presents CD105⁺/CD200⁺ expression in cell formulationscomprising dextran 40, or maltodextran, sucrose, trehalose, heparin,hetastarch or glycogen instead of dextran 40.

FIG. 22 presents BTR Data for dextran 40 and six non-dextran 40different sugars/polysaccharides.

FIG. 23 presents cellular aggregation measured by FRA acrossformulations comprising 10% human serum albumin (HSA), 10% bovine serumalbumin (BSA) or 10% fetal bovine serum (FBS). Assay control=100%dextran 40 solution, with cell stain, without cells.

FIG. 24: Cellular post-thaw viability across formulations comprising 10%HSA, 10% BSA or 10% FBS.

FIG. 25 presents post thaw recovery across formulations comprising 10%HSA, 4% HSA, 10% BSA or 10% FBS.

FIG. 26 presents cell identity measured by CD105⁺/CD200⁺ expressionacross formulations comprising 10% HSA, 4% HSA, 10% BSA or 10% FBS.

FIG. 27 presents CD34−/CD10+ expression across formulations comprising10% HSA, 10% BSA or 10% FBS.

FIG. 28 presents cell functionality measured by the BTR assay acrossformulations comprising 10% HSA, 4% HSA, 10% BSA or 10% FBS.

FIG. 29 presents FRA cellular aggregation results for bone marrowderived mesenchymal stem cells (BMMSC) cells and natural killer (NK)cells. Data are expressed in pixels per million cells loaded (px/MM).

5. DETAILED DESCRIPTION

5.1 Definitions

As used here, the term “about” means, e.g., within 10% of a statedfigure or value.

As used herein, “macro cell clump” means an aggregation of cells visiblewithout magnification, e.g., visible to the naked eye, and generallyrefers to a cell aggregation larger than about 150 microns.

As used herein, “micro cell clump” means a cell aggregation visible onlywith magnification, and generally refers to a cell aggregation smallerthan about 150 microns.

As used herein, the term “SH2” refers to an antibody that binds anepitope on the marker CD105. Thus, cells that are referred to as SH2⁺are CD105⁺.

As used herein, the terms “SH3” and SH4″ refer to antibodies that bindepitopes present on the marker CD73. Thus, cells that are referred to asSH3⁺ and/or SH4⁺ are CD73⁺.

As used herein, the term “isolated cell,” e.g., “isolated stem cell,”means a cell that is substantially separated from other cells of thetissue, e.g., placenta, from which the cell is derived. A cell is“isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% ofthe cells with which the cell is naturally associated, are removed fromthe cell, e.g., during collection and/or culture of the cell.

As used herein, “multipotent,” when referring to a cell, means that thecell has the ability to differentiate into some, but not necessarilyall, types of cells of the body, or into cells having characteristics ofsome, but not all, types of cells of the body. In certain embodiments,for example, an isolated multipotent cell that has the capacity todifferentiate into either of cells having characteristics ofchondrogenic or osteogenic cells is a multipotent cell.

As used herein, the term “population of isolated cells” means apopulation of cells that is substantially separated from other cells ofa tissue, e.g., placenta, from which the population of cells is derived.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from a mammalian placenta, regardless ofmorphology, cell surface markers, or the number of passages after aprimary culture. A placental stem cell is not obtained, and is notobtainable, from blood, e.g., cord blood or placental blood. The terms“placental stem cell” and “placental multipotent cell” as used herein donot, however, refer to, and placental stem cells and placentalmultipotent cells are not, trophoblasts, angioblasts, hemangioblasts,embryonic germ cells, embryonic stem cells, or cells obtained from theinner cell mass of a blastocyst, a cell obtained from an embryonicgonadal ridge, e.g., an embryonic germ cell. A cell is considered a“stem cell” if the cell displays attributes of a stem cell, e.g., amarker or gene expression profile associated with one or more types ofstem cells; the ability to replicate at least 10-40 times in culture,the ability to differentiate into cells of one or more of the three germlayers; the lack of adult (i.e., differentiated) cell characteristics,or the like. The terms “placental stem cell” and “placenta-derived stemcell” may be used interchangeably. Unless otherwise noted herein, theterm “placental” includes the umbilical cord. The placental stem cellsdisclosed herein, in certain embodiments, differentiate in vitro (underdifferentiating conditions), differentiate in vivo, or both.

As used herein, a cell, e.g., a stem cell, is “positive” for aparticular marker when that marker is detectable above background. Forexample, a placental stem cell is positive for, e.g., CD73 because CD73is detectable on placental stem cells in an amount detectably greaterthan background (in comparison to, e.g., an isotype control). A cell isalso positive for a marker when that marker can be used to distinguishthe cell from at least one other cell type, or can be used to select orisolate the cell when present or expressed by the cell. In the contextof, e.g., antibody-mediated detection, “positive,” as an indication aparticular cell surface marker is present, means that the marker isdetectable using an antibody, e.g., a fluorescently-labeled antibody,specific for that marker; “positive” also refers to a cell exhibitingthat marker in a amount that produces a signal, e.g., in a cytometer,that is detectably above background. For example, a cell is “CD200⁺”where the cell is detectably labeled with an antibody specific to CD200,and the signal from the antibody is detectably higher than that of acontrol (e.g., background or an isotype control). Conversely, “negative”in the same context means that the cell surface marker is not detectableusing an antibody specific for that marker compared to background. Forexample, a cell is “CD34⁻” where the cell is reproducibly not detectablylabeled with an antibody specific to CD34 to a greater degree than acontrol (e.g., background or an isotype control). Markers not detected,or not detectable, using antibodies are determined to be positive ornegative in a similar manner, using an appropriate control. For example,a cell or population of cells can be determined to be OCT-4⁺ if theamount of OCT-4 RNA detected in RNA from the cell or population of cellsis detectably greater than background as determined, e.g., by a methodof detecting RNA such as RT-PCR, slot blots, etc. Unless otherwise notedherein, cluster of differentiation (“CD”) markers are detected usingantibodies. OCT-4 can be determined to be present, and a cell is“OCT-4⁺”, if OCT-4 RNA is detectable using RT-PCR.

5.2 Improved Compositions Comprising Cells and Methods of Making theCompositions

Provided herein are improved methods of making compositions comprisingcells, e.g., stem cells, e.g., placental stem cells, and improvedcompositions, e.g., pharmaceutical compositions, produced thereby.Compositions, e.g., compositions administrable in liquid form,comprising cells are generally better tolerated by a recipient when,e.g., cell clumps, particularly cell clumps visible to the naked eye(i.e., macro clumps), are removed prior to administration of thepharmaceutical composition to an individual. The methods of makingcompositions comprising cells, e.g., stem cells, such as stem cells froma human postpartum placenta that has been drained of blood, placentalcells, as described herein result in compositions that are substantiallybetter tolerated when administered to an individual.

In one embodiment, provided herein is a method of making a composition,comprising filtering a solution comprising cells to produce a filteredcell-containing solution; diluting the filtered cell-containing solutionwith a first dilution solution to no more than about 10±3×10⁶ cells permilliliter, e.g., prior to cryopreservation; and optionally diluting theresulting filtered cell-containing solution with a second dilutionsolution comprising dextran to produce said composition. In anotherembodiment, provided herein is a method of making a composition,comprising filtering a solution comprising cells to produce a filteredcell-containing solution; diluting the filtered cell-containing solutionwith a first dilution solution to no more than about 1 to 50×10⁶, 1 to40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells permilliliter, e.g., prior to cryopreservation; and optionally diluting theresulting filtered cell-containing solution with a second dilutionsolution comprising dextran to produce said composition. In certainembodiments, if the number of cells is less than about 10±3×10⁶ cellsper milliliter, filtration is optional.

In a specific embodiment, the cells are stem cells. In a more specificembodiment, the stem cells are bone marrow-derived mesenchymal stemcells, or adult stem cells. In a specific embodiment, the cells areisolated placental cells. In a more specific embodiment, the isolatedplacental cells are placental stem cells or placental multipotent cells.In another specific embodiment, the cells are cells from placentalperfusate, e.g., nucleated cells from placental perfusate. Methods ofobtaining placental perfusate cells are described in Section 5.3.4,below.

In a specific embodiment, the cells are cryopreserved between saiddiluting with a first dilution solution and said diluting with saidsecond dilution solution. In another specific embodiment, the firstdilution solution comprises dextran and HSA. In another specificembodiment, said dextran in said first dilution solution is about 2.5%,2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%,5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%,8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% dextran. In anotherspecific embodiment, said dextran in said first dilution solution orsaid second dilution solution is dextran 1. In another specificembodiment, said dextran in said first dilution solution and said seconddilution solution is dextran 1. In another specific embodiment, saiddextran in said first dilution solution or said second dilution solutionis dextran 70. In another specific embodiment, said dextran in saidfirst dilution solution and said second dilution solution is dextran 70.In another specific embodiment, the dextran in said first dilutionsolution or said second dilution solution is dextran 40. In anotherspecific embodiment, the dextran in said first dilution solution andsaid second dilution solution is dextran 40. In another specificembodiment, said dextran 40 in said first dilution solution is about2.5% dextran 40 to about 10% dextran 40. In another specific embodiment,said dextran 40 in said first dilution solution is about 2.5%, 3.0%,3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 5.75%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%, 8.5%,9.0%, 9.5% or 10% dextran 40. In another specific embodiment, saiddextran 40 in said first dilution solution is about 5.5% dextran 40.

In other embodiments, said first and/or second dilution solutions maycomprise a polysaccharide in addition to or other than, i.e., in placeof, dextran. In certain embodiments, the polysaccharide is a polymer (2or more subunits) of glucose, and does not comprise saccharide subunitsthat are not glucose. In other embodiments, said first and/or seconddilution solutions comprise one or more of maltodextrin (e.g., about2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%,5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%,8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10%maltodextrin), trehalose (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% trehalose), or hetastarch (e.g., about 2.5%,2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%,5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%,8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% hetastarch). Inother embodiments, the first and/or second dilution solutions compriseone or more of sucrose (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% sucrose), heparin (e.g., about 55 USPunits/ml heparin), or glycogen (e.g., about 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10% glycogen). In a particular embodiment,said first and/or second dilution solutions comprises maltodextran inaddition to or other than, i.e., in place of, dextran. In anotherparticular embodiment, said first and/or second dilution solutionscomprises trehalose in addition to or other than, i.e., in place of,dextran. In another particular embodiment, said first and/or seconddilution solutions comprises hetastarch in addition to or other than,i.e., in place of, dextran.

In another specific embodiment, said HSA in said solution comprising HSAis about 1 to 17% HSA. In another specific embodiment, said HSA in saidsolution comprising HSA is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16% or about 17% HSA. In another specificembodiment, said HSA in said solution comprising HSA is about 4 to 10%HSA. In another specific embodiment, said HSA in said solutioncomprising HSA is about 3.125% HSA. In another specific embodiment, saidHSA in said solution comprising HSA is 5% HSA. In another specificembodiment, said HSA in said solution comprising HSA is 10% HSA. Inanother specific embodiment, said HSA in said solution comprising HSA isabout 16.875%% HSA. In another specific embodiment, said first dilutionsolution comprises HSA. In another specific embodiment, said HSA in saidfirst dilution solution is about 1 to 17% HSA. In another specificembodiment, said HSA in said first dilution solution is about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% or 17%HSA. In another specific embodiment, said HSA in said first dilutionsolution is about 4 to 10% HSA. In another specific embodiment, said HSAin said first dilution solution is about 3.125% HSA. In another specificembodiment, said HSA in said first dilution solution is about 5% HSA. Inanother specific embodiment, said HSA in said first dilution solution isabout 10% HSA. In another specific embodiment, said HSA in said firstdilution is about 16.875% HSA.

In other embodiments, bovine serum albumin (BSA)(e.g., about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% BSA) or fetalbovine serum (FBS) (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14% or 15% FBS) may be used in addition to or in placeof, i.e., instead of HSA in said solution.

In some embodiments, the ratio of HSA to dextran, e.g., dextran 1,dextran 40 or dextran 70, in the first solution is between about 6:1HSA:dextran to about 1:2.6 HSA:dextran. In some embodiments, the ratioof HSA to dextran is about 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1,2.5:1, 2.0:1, 1.5:1, 1:1, 1:1.5, 1:2 or 1:2.6 HSA:dextran. In someembodiments, the ratio of HSA to dextran, e.g., dextran 1, dextran 40 ordextran 70, in the first solution is about 3.13% HSA/8.25% dextran. Insome embodiments, the ratio of HSA to dextran, e.g., dextran 1, dextran40 or dextran 70, in the first solution is about 16.88% HSA/2.75%dextran. In particular embodiments, the ratio of HSA to dextran, e.g.,dextran 1, dextran 40 or dextran 70, in the first solution is about 10%HSA/5.5% dextran, e.g., dextran 1, dextran 40 or dextran 70.

In another specific embodiment, said first dilution solution furthercomprises a cryoprotectant. In a more specific embodiment, saidcryoprotectant is dimethylsulfoxide (DMSO). In a particular embodiment,said first dilution solution further comprises about 1% to about 15%,about 2.5% to about 15%, about 2.5% to about 10%, about 5% to about 15%,about 5% to about 10% or about 10% to about 15% DMSO. In a particularembodiment, said first dilution solution further comprises about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% DMSO. In aparticular embodiment, said first dilution solution further comprisesabout 5% DMSO.

In a specific embodiment, said first dilution solution comprises about5.5% dextran 40, about 10% HSA, and about 5% DMSO.

In another specific embodiment, said dextran 40 in said second dilutionsolution is about 10% dextran 40. In another specific embodiment, saidcomposition comprising cells comprises about 2.5%, 3.0%, 3.5%, 4.0%,4.5%, 5.0%, 5.5%, 5.75%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%, 8.5%, 9.0%, 9.5%or 10% dextran. In another specific embodiment, said compositioncomprising cells comprises about 7.5% to about 9% dextran. In anotherspecific embodiment, said composition comprises about 1.5×10⁶ cells permilliliter to about 5.0×10⁶ cells per milliliter. In another specificembodiment, said composition comprises about 1.0±0.3×10⁶ cells permilliliter to about 5.0±1.5×10⁶ cells per milliliter. In a specificembodiment, said second dilution solution does not comprise HSA.

The dextran usable in the methods provided herein can be dextran ofmolecular weight between about 1 kDa and about 150 kDa, e.g., 1 kDa(dextran 1), about 40 kDa (dextran 40) or about 70 kDa (dextran 70).

In another specific embodiment, the solution comprising cells comprisesa cryoprotectant. If the solution comprising cells comprises fewer thanabout 10±3×10⁶ cells per milliliter, the first dilution step can beomitted, and, in certain embodiments, the solution into which the cellsare suspended can comprise a cryopreservative, e.g., DMSO, e.g., about2% to about 15% DMSO, e.g., about 5% DMSO.

In another embodiment, provided herein is a method of making acomposition, comprising: (a) filtering a solution comprising cells,e.g., isolated placental cells, e.g., placental stem cells or placentalmultipotent cells, or cells isolated from placental perfusate, e.g.,total nucleated cells isolated from placental perfusate, dextran andhuman serum albumin (HSA) to produce a filtered cell-containingsolution; (b) optionally diluting said filtered cell-containing solutionto about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to15×10⁶, or 1 to 10×10⁶ cells per milliliter with a first dilutionsolution comprising dextran; and (c) optionally diluting the filteredcell-containing solution with a second dilution solution comprisingdextran but not comprising HSA, thereby making a composition. In someembodiments, step (b) is performed where the filtered cell-containingsolution in (a) comprises greater than about 15×10⁶ cells permilliliter, wherein said diluting in step (b) is to about 15×10⁶ cellsper milliliter. In some embodiments, step (b) is performed where thefiltered cell-containing solution in (a) comprises greater than about10±3×10⁶ cells per milliliter, wherein said diluting in step (b) is toabout 10±3×10⁶ cells per milliliter. In some embodiments in which saidfiltered cell-containing solution comprises less than about 10±3×10⁶cells per milliliter, diluting in step (b) is omitted and the solutionin step (a) comprises a cryoprotectant, e.g., DMSO, e.g., about 2% toabout 15% DMSO. In some embodiments, step (b) is performed where thefiltered cell-containing solution in (a) comprises greater than about7.5×10⁶ cells per milliliter, wherein said diluting in step (b) is toabout 7.5×10⁶ cells per milliliter. In a specific embodiment of themethod, said cells are cryopreserved prior to step (c). In certainembodiments, if the number of cells is less than about 10±3×10⁶ cellsper milliliter, filtration is optional. In a specific embodiment of themethod, said cells are cryopreserved prior to step (c). In anotherspecific embodiment, said dextran in said first dilution solution orsaid second dilution solution is dextran 40. In another specificembodiment, said dextran in said first dilution solution and said seconddilution solution is dextran 40. In another specific embodiment, saiddextran 40 in said first dilution solution is 5.0% dextran 40. Inanother specific embodiment, said dextran 40 in said first dilutionsolution is 5.5% dextran 40. In another specific embodiment, said HSA insaid solution comprising cells is about 1% HSA to about 15% HSA. Inanother specific embodiment, said first dilution solution comprises HSA.In a more specific embodiment, said HSA in said first dilution solutionis 5% HSA. In a more specific embodiment, said HSA in said firstdilution solution is 10% HSA. In another specific embodiment, said firstdilution solution further comprises a cryoprotectant. In a more specificembodiment, said cryoprotectant is DMSO, e.g., about 2% to about 15%DMSO. In another specific embodiment, said dextran 40 in said seconddilution solution is 10% dextran 40. In another specific embodiment,said solution in step (a) comprises a cryoprotectant.

In one aspect of the method, the number of cell clumps in the finalcomposition comprising cells is reduced or eliminated using filtration,preferably before cryopreservation. In certain embodiments in which thecells in the cell-containing solution are cryopreserved, thecell-containing solution is filtered prior to cryopreservation. Forexample, cells, e.g., placental stem cells, in solution can be passedthrough a filter prior to cryopreservation to remove visible cell clumps(aggregations of cells, i.e., macro cell clumps). In one embodiment,filtration comprises filtering the cell-containing solution through afilter prior to cryopreserving said cells, wherein said filter comprisespores between about 50 μM and about 150 μM in diameter (that is, thefilter is about a 50 μM filter to about a 150 μM filter), wherein thefilter is suitable for filtering solutions comprising cells. Forexample, the filter can be a filter comprising pores between about 50and about 80 μM, between about 60 μM and about 90 μM, between about 70μM and about 100 μM, between about 80 μM and about 110 μM, between about90 μM and about 120 μM, between about 100 μM and about 130 μM, betweenabout 110 μM and about 140 μM, or between about 120 μM and about 150 μM,or can be a 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145 or 150 μM filter. In a specific embodiment,said filter is a 70 μM filter. In another specific embodiment, saidfilter is a 100 μM filter. In another specific embodiment, said filteris about a 70 μM filter to a 100 μM filter. In another specificembodiment, said cell-containing solution is filtered after thawing inaddition to being filtered prior to freezing.

In other certain embodiments, if the number of cells is less than about10±3×10⁶ cells per milliliter, filtration is optional.

In various embodiments, the method of making a composition comprisingcells, e.g., isolated placental cells, comprises cryopreserving thecells at no more than about 50×10⁶, 40×10⁶, 30×10⁶, 20×10⁶, 15×10⁶,10×10⁶, 9.5×10⁶, 9×10⁶, 8.5×10⁶, 8×10⁶, 7.5×10⁶, 7×10⁶, 6.5×10⁶, 6×10⁶,5.5×10⁶, 5×10⁶, 4.5×10⁶, 4×10⁶, 3.5×10⁶, 3×10⁶, or 2.5×10⁶ cells permilliliter. In a specific embodiment, the cells are cryopreserved at nomore than about 10±3×10⁶ cells per milliliter. In another specificembodiment, the cells are cryopreserved at no more than about 15×10⁶cells per milliliter. In another specific embodiment, the cells arecryopreserved at no more than about 5×10⁶ cells per milliliter. Inanother specific embodiment, the cells are cryopreserved at about5.0×10⁶ to about 7.5×10⁶ cells per milliliter. In another specificembodiment, the cells are cryopreserved at about 5×10⁶ cells permilliliter. In another specific embodiment, the cells are cryopreservedat about 7.5×10⁶ cells per milliliter. In a specific embodiment, thecells are cryopreserved at about 10±3×10⁶ cells per milliliter. Inanother specific embodiment, said cells are cryopreserved at a numberthat, when said cells are thawed and diluted 1:1 to 1:11 (v/v), e.g.,1:1 to 1:5 (v/v), with dextran 40, e.g., 10% dextran 40, results in theformation of 2 or fewer visible cell clumps (i.e., macro cell clumps)per 10⁶ cells. In another specific embodiment, said isolated placentalcells are cryopreserved at a number that, when said cells are thawed anddiluted 1:1 to 1:11 (v/v), e.g., 1:1 to 1:5 (v/v), with dextran 40,e.g., 10% dextran 40, results in the formation of no visible cellclumps. In another specific embodiment, said cells are cryopreserved ata number that, when said cells are thawed and diluted 1:1 to 1:11 (v/v),e.g., 1:1 to 1:5 (v/v), with 10% dextran 40, results in the formation offewer than about 150, 140, 130, 120, 110 or 100 micro cell clumps per10⁶ cells.

In another embodiment, provided herein is a method of making acomposition, e.g., comprising contacting cells, e.g., isolated placentalcells, after cryopreservation with a solution comprising dextran 40,e.g., resuspending the cells or diluting the cells in a solutioncomprising dextran 40. In a specific embodiment, the solution comprisesbetween about 2.5% dextran 40 to about 10% dextran 40 (w/v). In specificembodiments, the solution comprises about 5% dextran 40 to about 10%dextran 40 (w/v). In another specific embodiment, the solution is a 5.0%dextran solution or a 10% dextran solution. In another specificembodiment, the solution is a 5.5% dextran solution or a 10% dextransolution. In other embodiments, the dextran has a molecular weight,e.g., an average molecular weight, between about 1 kilodaltons and about150 kilodaltons. In other embodiments, the dextran has a molecularweight, e.g., an average molecular weight, between about 1 kDa to about150 kDa, about 1 kDa to about 125 kDa, about 1 kDa to about 100 kDa,about 1 kDa to about 75 kDa, about 1 kDa to about 50 kDa, or about 1 kDato about 25 kDa. In other embodiments, the dextran has a molecularweight, e.g., an average molecular weight, between about 1 kDA to about10 kDa, about 30 kDa to about 50 kDa, or about 60 kDa to about 80 kDa.In other embodiments, the solution comprises between about 2% dextranand about 25% dextran. In a specific embodiment, said solution comprisesno HSA. In another specific embodiment, said solution is density matchedto said cells, e.g., said placental stem cells, e.g., the solution iswithin 5%, 2%, 1%, 0.5%, 0.2% or 0.1% of the density of the isolatedplacental cells. In another specific embodiment, the solution is notdensity-matched to said cells.

In another embodiment, the method of making a composition comprisingcells, e.g., isolated placental cells, comprises (a) filtering acell-containing solution comprising said cells prior tocryopreservation; (b) cryopreserving the cells at about 1 to 50×10⁶, 1to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cellsper milliliter; (c) thawing the cells; and (d) diluting thecell-containing solution about 1:1 (v/v) to about 1:11 with a dextran 40solution. In certain embodiments, about 15×10⁶ cells are cryopreservedin step (b). In certain embodiments, the cells are cryopreserved in step(b) at no more than 15×10⁶ cells per milliliter. In certain embodiments,no more than about 10±3×10⁶ cells per milliliter are cryopreserved instep (b). In other certain embodiments, if the number of cells is lessthan about 10±3×10⁶ cells per milliliter, filtration is optional. In amore specific embodiment, the cells in step (b) are cryopreserved in asolution comprising about 5% to about 10% dextran 40 and HSA.

In another embodiment, the method of making a composition comprises thefollowing steps:

(a) filtering a solution comprising cells, 5.5% dextran 40 solution, and10% HSA through a 70 μM filter to produce a filtered cell-containingsolution;

(b) diluting the filtered cell-containing solution with a solutioncomprising 5.5% dextran 40, 10% HSA, and 5% DMSO to about 1 to 50×10⁶, 1to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cellsper milliliter;

(d) cryopreserving the cells in said filtered cell-containing solution;

(e) thawing the cells; and

(f) optionally diluting the filtered cell-containing solution with 10%dextran 40.

In certain embodiments, said diluting in step (b) is to no more thanabout 15×10⁶ cells per milliliter. In certain embodiments, said dilutingin step (b) is to no more than about 10±3×10⁶ cells/mL. In embodimentsin which the filtered cell-containing solution comprises fewer thanabout 10±3×10⁶ cells/mL, the solution in step (a) comprises acryoprotectant, e.g., DMSO, e.g., about 1% to about 5% DMSO, and step(b) is omitted. In other certain embodiments, if the number of cells isless than about 10±3×10⁶ cells per milliliter, filtration is optional.In some embodiments, step (f) comprises diluting the filteredcell-containing solution 1:1 to 1:11 (v/v) with 10% dextran 40. In someembodiments, step (f) comprises diluting the filtered cell-containingsolution 1:1 to 1:5 (v/v) with 10% dextran 40.

In another embodiment, the method of making a composition providedherein comprises the following steps:

(a) centrifuging a plurality of cells to collect the cells;

(b) resuspending the cells in 5.5% dextran 40;

(c) centrifuging the cells to collect the cells;

(d) resuspending the cells in a 5.5% dextran 40 solution that comprises10% HSA to produce a cell-containing solution;

(e) filtering the cell-containing solution through a 70 μM filter toproduce a filtered cell-containing solution;

(f) diluting the filtered cell-containing solution in 5.5% dextran 40,10% HSA, and 5% DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter;

(g) cryopreserving the cells in said filtered cell-containing solution;

(h) thawing the cells; and

(i) optionally diluting the filtered cell-containing solution with 10%dextran 40.

In certain embodiments, said diluting in step (f) is to no more thanabout 15×10⁶ cells per milliliter. In certain embodiments, said dilutingin step (t) is no more than about 10±3×10⁶ cells/mL. In other certainembodiments, if the number of cells is less than about 10±3×10⁶ cellsper milliliter, filtration is optional. In embodiments in which saidresuspending in step (d) produces a cell-containing solution comprisingfewer than about 10±3×10⁶ cells/mL, the solution in step (d) comprises acryoprotectant, e.g., DMSO, e.g., about 1% to about 5% DMSO, and step(t) is omitted. In some embodiments, step (i) comprises diluting thefiltered cell-containing solution 1:1 to 1:5 (v/v) with 10% dextran 40.In some embodiments, step (i) comprises diluting the filteredcell-containing solution 1:1 to 1:11 (v/v) with 10% dextran 40.

In a specific embodiment of any of the above methods, DMSO issubstantially removed from the composition comprising cells, such thatthe final concentration of DMSO in the composition is less than about2.5%, 2.0%, 1.5%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%or about 0.1%. Removal of DMSO can be accomplished, e.g., bycentrifuging the cells and resuspending the cells in 10% dextran 40.Such a centrifuging and resuspending step can be repeated one or moretimes.

In another specific embodiment of any of the above methods, the methodfurther comprises concentrating the resulting cell composition to about5×10⁶ cells per milliliter to 1×10⁸ cells per milliliter. Such acomposition is useful, for example, for subcutaneous administration ofthe composition to an individual in need thereof.

In another specific embodiment of any of the above methods, the cell isa cell other than a placental stem cell. In more specific embodiments,for example, the cells can be stem cells or non-stem cells. In specificembodiments in which the cells are stem cells, the stem cells may be,e.g., adult stein cells, somatic stem cells, embryonic stem cells,embryonic germ cells, umbilical cord stem cells, amniotic fluid stemcells, bone marrow-derived mesenchymal stem cells, cord blood-derivedmesenchymal stem cells, peripheral blood-derived mesenchymal stem cells,adipose-derived mesenchymal stem cells or periosteum-derived stem cells.In another specific embodiment, the cells are natural killer cells,e.g., CD3⁻, CD56⁺ natural killer cells.

In another aspect, provided herein are compositions, e.g.,pharmaceutical compositions. In certain embodiments, the compositionsare made by the above methods. In certain embodiments, the compositionslack visible cell clumps, i.e., macro cell clumps. In certain otherembodiments, the compositions comprise substantially reduced numbers ofmicro cell clumps (those visible only with a microscope, e.g., a lightmicroscope) compared to compositions that have not been filtered, e.g.,about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% fewer micro cellclumps.

In one embodiment, provided herein is a composition, e.g., apharmaceutical composition, comprising a plurality of cells, e.g., aplurality of isolated placental cells, or cells isolated from placentalperfusate, e.g., total nucleated cells from placental perfusate, in asolution comprising 10% dextran 40, wherein said composition comprisesbetween about 1.0±0.3×10⁶ cells per milliliter to about 5.0±1.5×10⁶cells per milliliter, and wherein said composition comprises no visiblecell clumps (i.e., comprises no macro cell clumps). In some embodiments,said composition comprises between about 1.5×10⁶ cells per milliliter toabout 3.75×10⁶ cells per milliliter. In certain other embodiments, thecomposition comprises between about 1.0×10⁶ cells per milliliter and15×10⁶ cells per milliliter, e.g., between about 7.5×10⁶ cells permilliliter and about 15×10⁶ cells per milliliter. In certain otherembodiments, the composition comprises less than about 20×10⁶ cells permilliliter. In a specific embodiment, said cells have been cryopreservedand thawed. In another specific embodiment, said cells have beenfiltered through a 70 μM to 100 μM filter. In another specificembodiment, said composition comprises no macro cell clumps. In anotherspecific embodiment, said composition comprises fewer than about 200micro cell clumps per 10⁶ cells. In another specific embodiment, saidcomposition comprises fewer than about 150 micro cell clumps per 10⁶cells. In another specific embodiment, said composition comprises fewerthan about 100 micro cell clumps per 10⁶ cells. In another specificembodiment, said composition comprises less than 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% DMSO.

In some embodiments, the composition comprises about 2.5%, 2.75%, 3.0%,3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%,6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%,8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% dextran, e.g., dextran 1,dextran 40 or dextran 70. In a specific embodiment, said compositioncomprises about 7.5% to about 9% dextran 40. In a specific embodiment,said composition comprises about 5.5% dextran 40.

In other embodiments, said composition comprises a polysaccharide inaddition to or other than, i.e., in place of, dextran. In certainembodiments, the polysaccharide is a polymer of glucose that does notcomprise non-glucose saccharide subunits. In other embodiments, saidcomposition comprises maltodextrin (e.g., about 2.5%, 2.75%, 3.0%,3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%,6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%,8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10% maltodextrin), trehalose (e.g.,about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%,5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%,7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10%trehalose), or hetastarch (e.g., about 2.5%, 2.75%, 3.0%, 3.25%, 3.5%,3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%,6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%,9.25%, 9.5%, 9.75%, or 10% hetastarch). In other embodiments, saidcomposition comprises sucrose (e.g., about 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10% sucrose), heparin (e.g., 55 USPunits/ml heparin), or glycogen (e.g., about 2.5%, 2.75%, 3.0%, 3.25%,3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%,6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%,9.0%, 9.25%, 9.5%, 9.75%, or 10% glycogen). In a particular embodiment,said composition comprises maltodextran in addition to or other than,i.e., in place of, dextran. In another particular embodiment, saidcomposition comprises trehalose in addition to or instead of dextran. Inanother particular embodiment, said composition comprises hetastarch inaddition to or instead of dextran.

In another specific embodiment, said composition comprises about 1% toabout 17% HSA. In some embodiments, said composition comprises about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, orabout 17% HSA. In some embodiments, said composition comprises about3.125% HSA. In some embodiments, said composition comprises about 5%HSA. In some embodiments, said composition comprises about 10% HSA. Insome embodiments, said composition comprises about 16.875% HSA.

In other embodiments, said composition comprises bovine serum albumin(BSA) (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14% or 15% BSA) or fetal bovine serum (FBS) (e.g., about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% FBS) inaddition to or instead of HSA.

In some embodiments, the composition comprises a cryoprotectant, e.g.,DMSO, e.g., about 1% to about 15% DMSO. In some embodiments, saidcomposition comprises about 1% to about 5% DMSO. In some embodiments,said composition comprises about 1% to about 15%, about 2.5% to about15%, about 2.5% to about 10%, about 5% to about 15%, about 5% to about10% or about 10% to about 15% DMSO. In some embodiments, saidcomposition comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14% or 15% DMSO. In a particular embodiment, thecomposition comprises about 5% DMSO.

Further provided herein are compositions comprising cells, wherein saidcompositions are produced by one of the methods disclosed herein. Forexample, in one embodiment, provided herein is a composition comprisingcells, wherein said composition is produced by a method comprisingfiltering a solution comprising the cells to form a filteredcell-containing solution; diluting the filtered cell-containing solutionwith a first solution to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter, e.g., priorto cryopreservation; and diluting the resulting filtered cell-containingsolution with a second solution comprising dextran but not comprisingHSA to produce said composition. In certain embodiments, said dilutingis to no more than about 15×10⁶ cells per milliliter. In certainembodiments, said diluting is to no more than about 10+3×10⁶ cells permilliliter. In certain embodiments, said diluting is to no more thanabout 7.5×10⁶ cells per milliliter. In other certain embodiments, if thenumber of cells is less than about 10±3×10⁶ cells per milliliter,filtration is optional. In a specific embodiment, the cells arecryopreserved between said diluting with a first dilution solution andsaid diluting with said second dilution solution. In another specificembodiment, the first dilution solution comprises dextran and HSA. Inanother specific embodiment, the dextran in said first dilution solutionor said second dilution solution is dextran 40. In another specificembodiment, the dextran in said first dilution solution and said seconddilution solution is dextran 40. In another specific embodiment, saiddextran 40 in said first dilution solution is 5.0% dextran 40. Inanother specific embodiment, said dextran 40 in said first dilutionsolution is 5.5% dextran 40. In another specific embodiment, said HSA insaid solution comprising HSA is 10% HSA. In another specific embodiment,said first dilution solution comprises HSA. In a more specificembodiment, said HSA in said first dilution solution is 10% HSA. Inanother specific embodiment, said first dilution solution comprises acryoprotectant. In a more specific embodiment, said cryoprotectant isDMSO. In another specific embodiment, said dextran 40 in said seconddilution solution is about 10% dextran 40. In another specificembodiment, said composition comprising cells comprises about 7.5% toabout 9% dextran. In another specific embodiment, said compositioncomprising cells comprises about 1.0±0.3×10⁶ cells per milliliter toabout 5.0±1.5×10⁶ cells per milliliter. In another specific embodiment,said composition comprising cells comprises about 1.5×10⁶ cells permilliliter to about 3.75×10⁶ cells per milliliter.

In another embodiment, the composition comprising cells is made by amethod comprising (a) filtering a cell-containing solution comprisingsaid cells prior to cryopreservation to produce a filteredcell-containing solution; (b) cryopreserving the cells in the filteredcell-containing solution at about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶,1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter; (c)thawing the cells; and (d) diluting the filtered cell-containingsolution about 1:1 to about 1:11 (v/v) with a dextran 40 solution. Incertain embodiments, if the number of cells is less than about 10±3×10⁶cells per milliliter, filtration is optional. In a more specificembodiment, the cells in step (b) are cryopreserved at about 10±3×10⁶cells per milliliter. In a more specific embodiment, the cells in step(b) are cryopreserved in a solution comprising about 5% to about 10%dextran 40 and HSA. In certain embodiments, said diluting in step (d) isto no more than about 15×10⁶ cells per milliliter.

In another embodiment, the composition comprising cells is made by amethod comprising: (a) suspending the cells in a 5.5% dextran 40solution that comprises 10% HSA to form a cell-containing solution; (b)filtering the cell-containing solution through a 70 μM filter; (c)diluting the cell-containing solution with a solution comprising 5.5%dextran 40, 10% HSA, and 5% DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to30×10⁶, 1 to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter;(d) cryopreserving the cells; (e) thawing the cells; and (f) dilutingthe cell-containing solution 1:1 to 1:11 (v/v) with 10% dextran 40. Incertain embodiments, said diluting in step (b) is to no more than about15×10⁶ cells per milliliter. In certain embodiments, said diluting instep (b) is to no more than about 10±3×10⁶ cells/mL. In anotherembodiment, the composition comprising cells is made by a methodcomprising: (a) centrifuging a plurality of cells to collect the cells;(b) resuspending the cells in 5.5% dextran 40; (c) centrifuging thecells to collect the cells; (d) resuspending the cells in a 5.5% dextran40 solution that comprises 10% HSA; (e) filtering the cells through a 70μM filter; (f) diluting the cells in 5.5% dextran 40, 10% HSA, and 5%DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to15×10⁶, or 1 to 10×10⁶ cells per milliliter; (g) cryopreserving thecells; (h) thawing the cells; and (i) diluting the cells 1:1 to 1:11(v/v) with 10% dextran 40. In certain embodiments, said diluting in step(f) is to no more than about 15×10⁶ cells per milliliter. In certainembodiments, said diluting in step (f) is to no more than about 10±3×10⁶cells/mL. In other certain embodiments, if the number of cells is lessthan about 10±3×10⁶ cells per milliliter, filtration is optional.

The compositions, e.g., pharmaceutical compositions, comprising cellsdescribed herein can comprise any mammalian cell, including mammalianstem cells and mammalian non-stem cells. In some embodiments, thecompositions, e.g., pharmaceutical compositions, comprising cellsdescribed herein can comprise isolated placental cells, e.g., any of theisolated placental cells described herein (see, e.g., Section 5.3,below). In a specific embodiment, the isolated placental cells areCD34⁻, CD10⁺ and CD105⁺ as detected by flow cytometry. In a morespecific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cellsare placental stem cells. In another more specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ placental cells are multipotent placentalcells. In another specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺placental cells have the potential to differentiate into cells of aneural phenotype, cells of an osteogenic phenotype, or cells of achondrogenic phenotype. In a more specific embodiment, the isolatedCD34⁻, CD10⁺, CD105⁺ placental cells are additionally CD200⁺. In anothermore specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells are additionally CD90⁺ or CD45⁻, as detected by flow cytometry. Inanother more specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺placental cells are additionally CD90⁺ or CD45⁻, as detected by flowcytometry. In a more specific embodiment, the CD34⁻, CD10⁺, CD105⁺,CD200⁺ placental cells are additionally CD90⁺ or CD45⁻, as detected byflow cytometry. In another more specific embodiment, the CD34⁻, CD10⁺,CD105⁺, CD200⁺ cells are additionally CD90⁺ and CD45⁻, as detected byflow cytometry. In another more specific embodiment, the CD34⁻, CD10⁺,CD105⁺, CD200⁺, CD90⁺, CD45⁻ cells are additionally CD80⁻ and CD86⁻, asdetected by flow cytometry.

In a more specific embodiment, the CD34⁻, CD10⁺, CD105⁺ cells areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, CD80⁻, CD86⁻,SH3⁺ or SH4⁺. In another more specific embodiment, the cells areadditionally CD44⁺. In a specific embodiment of any of the isolatedCD34⁻, CD10⁺, CD105⁺ placental cells above, the cells are additionallyone or more of CD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺,HLA-DP,DQ,DR⁻, and/or Programmed Death-1 Ligand (PDL1)⁺.

In certain other embodiments of the compositions, said isolatedplacental cells are CD200⁺ and HLA-G⁺; CD73⁺, CD105⁺, and CD200⁺; CD200⁺and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺ and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells comprising said isolated placental cells when saidpopulation is cultured under conditions that allow the formation of anembryoid-like body; or OCT-4⁺ and facilitate the formation of one ormore embryoid-like bodies in a population of placental cells comprisingthe isolated placental cells when said population is cultured underconditions that allow formation of embryoid-like bodies; or anycombination thereof. In a specific embodiment, said CD200⁺, HLA-G⁺ cellsare CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specificembodiment, said CD73⁺, CD105⁺, and CD200⁺ cells are CD34⁻, CD38⁻,CD45⁻, and HLA-G⁺. In another specific embodiment, said CD200⁺, OCT-4⁺cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In anotherspecific embodiment, said CD73⁺, CD105⁺ and HLA-G⁺ cells are CD34⁻,CD45⁻, OCT-4⁺ and CD200⁺. In another specific embodiment, said CD73⁺ andCD105⁺ cells are OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said OCT-4⁺ cells are CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻,and CD45⁻. Isolated placental cells that can be contained within thecompositions comprising cells, described herein, are described in moredetail in Section 5.3, below.

In other specific embodiments of the compositions provided herein, thecell is a cell other than a placental stem cell. In more specificembodiments, for example, the cells can be stem cells or non-stem cells.In specific embodiments in which the cells are stem cells, the stemcells may be, e.g., adult stem cells, somatic stem cells, embryonic stemcells, embryonic germ cells, umbilical cord stem cells, amniotic fluidstem cells, bone marrow-derived mesenchymal stem cells, cordblood-derived mesenchymal stem cells, peripheral blood-derivedmesenchymal stem cells, adipose-derived mesenchymal stem cells orperiosteum-derived stem cells. In another specific embodiment, the cellsare natural killer cells, e.g., CD3⁻, CD56⁺ natural killer cells.

5.3 Isolated Placental Cells and Isolated Placental Cell Populations

The isolated placental multipotent cells useful in the compositions,e.g., pharmaceutical compositions, provided herein are obtainable from aplacenta or part thereof, that adhere to a tissue culture substrate andhave characteristics of multipotent cells or stem cells. In certainembodiments, the isolated placental cells useful in the methodsdisclosed herein have the capacity to differentiate into non-placentalcell types. The isolated placental cells useful in the methods disclosedherein can be either fetal or maternal in origin (that is, can have thegenotype of either the fetus or mother, respectively). Preferably, theisolated placental cells and populations of isolated placental cells arefetal in origin. As used herein, the phrase “fetal in origin” or“non-maternal in origin” indicates that the isolated placental cells orpopulations of isolated placental cells are obtained from the umbilicalcord or placental structures associated with the fetus, i.e., that havethe fetal genotype. As used herein, the phrase “maternal in origin”indicates that the cells or populations of cells are obtained from aplacental structures associated with the mother, e.g., which have thematernal genotype. Isolated placental cells, or populations of cellscomprising the isolated placental cells, can comprise isolated placentalcells that are solely fetal or maternal in origin, or can comprise amixed population of isolated placental cells of both fetal and maternalorigin. The isolated placental cells, and populations of cellscomprising the isolated placental cells, can be identified and selectedby the morphological, marker, and culture characteristics discussedbelow. Isolated placental cells suitable for use in the methods andcompositions described herein can include, for example, those describedin U.S. Patent Application Publication No. 2007/0275362 and U.S. Pat.No. 7,468,276, the disclosures of which are hereby incorporated byreference in their entireties.

5.3.1 Physical and Morphological Characteristics

The isolated placental cells described herein, when cultured in primarycultures or in cell culture, adhere to the tissue culture substrate,e.g., tissue culture container surface (e.g., tissue culture plastic),or to a tissue culture surface coated with extracellular matrix orligands such as laminin, collagen (e.g., native or denatured), gelatin,fibronectin, ornithine, vitronectin, and extracellular membrane protein(e.g., MATRIGEL®). (BD Discovery Labware, Bedford, Mass.). The isolatedplacental cells in culture assume a generally fibroblastoid, stellateappearance, with a number of cytoplasmic processes extending from thecentral cell body. The cells are, however, morphologicallydistinguishable from fibroblasts cultured under the same conditions, asthe isolated placental cells exhibit a greater number of such processesthan do fibroblasts. Morphologically, isolated placental cells are alsodistinguishable from hematopoietic stem cells, which generally assume amore rounded, or cobblestone, morphology in culture.

5.3.2 Cell Surface, Molecular and Genetic Markers

Isolated placental cells, e.g., multipotent cells or stem cells, andpopulations of isolated placental cells, express a plurality of markersthat can be used to identify and/or isolate the stem cells, orpopulations of cells that comprise the stem cells. The isolatedplacental cells, and placental cell populations described herein (thatis, two or more isolated placental cells) include placental cells andplacental cell-containing cell populations obtained directly from theplacenta, or any part thereof (e.g., amnion, chorion, placentalcotyledons, and the like). Isolated placental cell populations alsoinclude populations of (that is, two or more) isolated placental cellsin culture, and a population in a container, e.g., a bag. The isolatedplacental cells described herein are not trophoblasts, cytotrophoblasts,hemangioblasts, embryonic germ cells or embryonic stem cells. Placentalmultipotent cells usable in the methods and compositions describedherein are described in U.S. Patent Application Publication No.2007/0275362, and U.S. Pat. Nos. 7,045,148 and 7,468,276, thedisclosures of which are hereby incorporated by reference in theirentireties, and as described below.

The isolated placental cells, usable in the compositions and methodsprovided herein, generally express the markers CD73, CD105, CD200,HLA-G, and/or OCT-4, and do not express CD34, CD38, or CD45, and areHLA-DP, DQ, and DR⁻. The isolated multipotent cells are also generallyCD10⁺, CD29⁺, CD54⁺, CD90⁺, CD44⁺ and CD38⁺. In certain embodiments, thecells are one or more of SSEA3-, SSEA4- or ABC-p⁺. The isolatedplacental cells can also express HLA-ABC (MHC-1). These markers can beused, in any combination, to identify the isolated placental cells,e.g., isolated placental stem cells or isolated multipotent cells and todistinguish the isolated placental cells from other cell types. Becausethe isolated placental cells can express CD73 and CD105, they can havemesenchymal stem cell-like characteristics. Lack of expression of CD34⁻,CD38 and/or CD45, for example, identifies the isolated placental cellsas non-hematopoietic stem cells.

In certain embodiments, the isolated placental cells are isolatedplacental stem cells. In certain other embodiments, the isolatedplacental cells are isolated placental multipotent cells. In oneembodiment, the isolated placental cells are CD34⁻, CD10⁺ and CD105⁺ asdetected by flow cytometry. In a specific embodiment, the isolatedCD34⁻, CD10⁺, CD105⁺ placental cells are placental stem cells. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells are multipotent placental cells. In another specific embodiment,the isolated CD34⁻, CD10⁺, CD105⁺ placental cells have the potential todifferentiate into cells of a neural phenotype, cells of an osteogenicphenotype, or cells of a chondrogenic phenotype. In another specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells areadditionally CD200⁺. In another specific embodiment, the isolated CD34⁻,CD10⁺, CD105⁺ placental cells are additionally CD45⁻ or CD90⁺. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells are additionally CD45⁻ and CD90⁺, as detected by flow cytometry.In a more specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺placental cells are additionally CD90⁺ or CD45⁻, as detected by flowcytometry. In another more specific embodiment, the isolated CD34⁻,CD10⁺, CD105⁺, CD200⁺ placental cells are additionally CD90⁺ and CD45⁻,as detected by flow cytometry, i.e., the cells are CD34⁻, CD10⁺, CD45⁻,CD90⁺, CD105⁺ and CD200⁺. In a more specific embodiment, said CD34⁻,CD10⁺, CD45⁻, CD90⁺, CD105⁺, CD200⁺ cells are additionally CD80⁻ andCD86⁻.

In a specific embodiment, any of the CD34⁻, CD10⁺, CD105⁺ cellsdescribed above are additionally one or more of CD29⁺, CD38⁻, CD44⁺,CD54⁺, SH3⁺ or SH4⁺. In another more specific embodiment, the cells areadditionally CD44⁺. In another specific embodiment of any of theisolated CD34⁻, CD10⁺, CD105⁺ placental cells above, the cells areadditionally one or more of CD117⁺, CD133⁻, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺,HLA-DP,DQ,DR⁻, and/or PDL1⁺. In another specific embodiment, the CD34⁻,CD10⁺, CD105⁺ placental cells are additionally one or more of CD13⁺,CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺(CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺,CD117⁺, CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺,SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻,HLA-G⁺, or Programmed Death-1 Ligand (PDL1)⁺, or any combinationthereof. In a more specific embodiment, the CD34⁻, CD10⁺, CD105⁺placental cells are additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺,CD45⁻, CD54/ICAM⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺),CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁺, andProgrammed Death-1 Ligand (PDL1)⁺.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, CD34⁻, CD10⁺ and CD105⁺ placental cells. In variousembodiments, at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, or at least about 60% of cellsin said cell population are CD34⁻, CD10⁺ and CD105⁺ placental cells.Preferably, at least about 70% of cells in said cell population areCD34⁻, CD10 and CD105⁺ placental cells. More preferably, at least about90%, 95%, or 99% of said cells are CD34⁻, CD10⁺ and CD105⁺ placentalcells. In a specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺placental cells are additionally CD200⁺. In a more specific embodiment,the isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells areadditionally CD90⁺ or CD45⁻, as detected by flow cytometry. In anothermore specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺placental cells are additionally CD90⁺ and CD45⁻, as detected by flowcytometry. In a more specific embodiment, any of the isolated CD34⁻,CD10⁺, CD105⁺ placental cells described above are additionally one ormore of CD29⁺, CD38⁻, CD44⁺, CD54⁺, SH3⁺ or SH4⁺. In another morespecific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells,or isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells, areadditionally CD44⁺. In a specific embodiment of any of the populationsof cells comprising isolated CD34⁻, CD10⁺, CD105⁺ placental cells above,the isolated placental cells are additionally one or more of CD13⁺,CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁴(CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺,CD117⁻, CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺,SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻,HLA-G⁺, or Programmed Death-1 Ligand (PDL1)⁺, or any combinationthereof. In a more specific embodiment, the CD34⁻, CD10⁺, CD105⁺ cellsare additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54/ICAM⁺,CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺,SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁺, CD144/VE-cadherin^(low),CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻(VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁺, and Programmed Death-1Ligand (PDL1)⁺.

In certain embodiments, the isolated placental cells are one or more, orall, of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, and ABC-p⁺, wherein said isolatedplacental cells are obtained by physical and/or enzymatic disruption ofplacental tissue. In a specific embodiment, the isolated placental cellsare OCT-4⁺ and ABC-p⁺. In another specific embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, wherein said isolated placentalcells have at least one of the following characteristics: CD10⁺, CD29⁺,CD44⁺, CD45⁻, CD54⁺, CD90⁻, SH3⁺, SH4⁺, SSEA3⁻, and SSEA4⁻. In anotherspecific embodiment, the isolated placental cells are OCT-4⁺, CD34⁻,CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH3⁺, SH4⁺, SSEA3⁻, andSSEA4⁻. In another embodiment, the isolated placental cells are OCT-4⁺,CD34⁻, SSEA3⁻, and SSEA4⁻. In a more specific embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, and is either SH2⁺ or SH3⁺. In amore specific embodiment, the isolated placental cells are OCT-4⁺,CD34⁻, SH2⁺, and SH3⁺. In another more specific embodiment, the isolatedplacental cells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻, and are eitherSH2⁺ or SH3⁺. In another more specific embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, and either SH2⁺ or SH3⁺, and is atleast one of CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, orSSEA4⁻. In another more specific embodiment, the isolated placentalcells are OCT-4⁺, CD34⁻, CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺,SSEA3⁻, and SSEA4⁻, and either SH2⁺ or SH3⁺.

In one embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are CD200⁺ or HLA-G⁺. In aspecific embodiment, the isolated placental cells are CD200⁺ and HLA-G⁺.In another specific embodiment, the isolated placental cells are CD73⁺and CD105⁺. In another specific embodiment, said the isolated placentalcells are CD34⁻, CD38 or CD45⁻. In another specific embodiment, theisolated placental cells are CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, the isolated placental cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺and CD105⁺. In another specific embodiment, said CD200⁺ or HLA-G⁺isolated placental cells facilitate the formation of embryoid-likebodies in a population of placental cells comprising the isolatedplacental cells, under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, the isolatedplacental cells are isolated away from placental cells that are not stemor multipotent cells. In another specific embodiment, said isolatedplacental cells are isolated away from placental stem cells that do notdisplay these markers.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, CD200⁺, HLA-G⁺ placental cells. In various embodiments,at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, or at least about 60% of cells in saidcell population are CD200⁺, HLA-G⁺ placental cells. Preferably, at leastabout 70% of cells in said cell population are CD200⁺, HLA-G⁺ placentalcells. More preferably, at least about 90%, 95%, or 99% of said cellsare CD200⁺, HLA-G⁺ placental cells. In a specific embodiment of theisolated populations, said placental cells are also CD73⁺ and CD105⁺. Inanother specific embodiment, said placental cells are also CD34⁻, CD38⁻or CD45⁻. In a more specific embodiment, said placental cells are alsoCD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another embodiment, saidisolated population of cells produces one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies. In another specific embodiment, said population of placentalcells is isolated away from placental cells that are not stem cells. Inanother specific embodiment, said population of placental cells isisolated away from placental cells that do not display these markers.

In another embodiment, placental cells, usable in the compositions andmethods provided herein, are CD73⁺, CD105⁺, and CD200⁺. In anotherspecific embodiment, said placental cell is HLA-G⁺. In another specificembodiment, said placental cells are CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said placental cells are CD34⁻, CD38⁻ and CD45⁻. Ina more specific embodiment, said placental cells are CD34⁻, CD38⁻,CD45⁻, and HLA-G⁺. In another specific embodiment, the isolated CD73⁺,CD105⁺, and CD200⁺ placental cells facilitate the formation of one ormore embryoid-like bodies in a population of said placental cells, whenthe population is cultured under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, said placentalcells are isolated away from placental cells that are not stem cells. Inanother specific embodiment, said placental cells are isolated away fromplacental cells that do not display these markers.

In another embodiment, a cell population usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, isolated CD73⁺, CD105⁺, CD200⁺ placental cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said cell population are isolated CD73⁺, CD105⁺, CD200⁺placental cells. In another embodiment, at least about 70% of said cellsin said population of cells are isolated CD73⁺, CD105⁺, CD200⁺ placentalcells. In another embodiment, at least about 90%, 95% or 99% of cells insaid population of cells are isolated CD73⁺, CD105⁺, CD200⁺ placentalcells. In a specific embodiment of said populations, the isolatedplacental cells are HLA-G⁺. In another specific embodiment, the isolatedplacental cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, the isolated placental cells are additionallyCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, the isolatedplacental cells are additionally CD34⁻, CD38⁻, CD45⁻, and HLA-G′. Inanother specific embodiment, said population of cells produces one ormore embryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidpopulation of placental cells is isolated away from placental cells thatare not stem cells. In another specific embodiment, said population ofplacental stem cells is isolated away from placental cells that do notdisplay these characteristics.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are CD200⁺ and OCT-4⁺. In aspecific embodiment, the isolated placental cells are CD73⁺ and CD105⁺.In another specific embodiment, said isolated placental cells areHLA-G⁺. In another specific embodiment, said isolated CD200⁺, OCT-4⁺placental cells are CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said isolated CD200⁺, OCT-4⁺ placental cells are CD34⁻, CD38and CD45⁻. In a more specific embodiment, said isolated CD200⁺, OCT-4⁺placental cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. Inanother specific embodiment, the isolated CD200⁺, OCT-4⁺ placental cellsfacilitate the production of one or more embryoid-like bodies by apopulation of placental cells that comprises the isolated cells, whenthe population is cultured under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, said isolatedCD200⁺, OCT-4⁺ placental cells are isolated away from placental cellsthat are not stem cells. In another specific embodiment, said isolatedCD200⁺, OCT-4⁺ placental cells are isolated away from placental cellsthat do not display these characteristics.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, CD200⁺, OCT-4⁺ placental cells. In various embodiments,at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, or at least about 60% of cells in saidcell population are isolated CD200⁺, OCT-4⁺ placental cells. In anotherembodiment, at least about 70% of said cells are said isolated CD200⁺,OCT-4⁺ placental cells. In another embodiment, at least about 80%, 90%,95%, or 99% of cells in said cell population are said isolated CD200⁺,OCT-4⁺ placental cells. In a specific embodiment of the isolatedpopulations, said isolated CD200⁺, OCT-4⁺ placental cells areadditionally CD73⁺ and CD105⁺. In another specific embodiment, saidisolated CD200⁺, OCT-4⁺ placental cells are additionally HLA-G⁺. Inanother specific embodiment, said isolated CD200⁺, OCT-4⁺ placentalcells are additionally CD34⁻, CD38⁻ and CD45⁻. In a more specificembodiment, said isolated CD200⁺, OCT-4⁺ placental cells areadditionally CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In anotherspecific embodiment, the cell population produces one or moreembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidcell population is isolated away from placental cells that are notisolated CD200⁺, OCT-4⁺ placental cells. In another specific embodiment,said cell population is isolated away from placental cells that do notdisplay these markers.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are CD73⁺, CD105⁺ and HLA-G⁺.In another specific embodiment, the isolated CD73⁺, CD105⁺ and HLA-G⁺placental cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, the isolated CD73⁺, CD105⁺, HLA-G⁺ placental cellsare additionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,the isolated CD73⁺, CD105⁺, HLA-G⁺ placental cells are additionallyOCT-4⁺. In another specific embodiment, the isolated CD73⁺, CD105⁺,HLA-G⁺ placental cells are additionally CD200⁺. In a more specificembodiment, the isolated CD73⁺, CD105⁺, HLA-G⁺ placental cells areadditionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specificembodiment, the isolated CD73⁺, CD105⁺, HLA-G⁺ placental cellsfacilitate the formation of embryoid-like bodies in a population ofplacental cells comprising said cells, when the population is culturedunder conditions that allow the formation of embryoid-like bodies. Inanother specific embodiment, said the isolated CD73⁺, CD105⁺, HLA-G⁺placental cells are isolated away from placental cells that are not theisolated CD73⁺, CD105⁺, HLA-G⁺ placental cells. In another specificembodiment, said the isolated CD73⁺, CD105⁺, HLA-G⁺ placental cells areisolated away from placental cells that do not display these markers.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, isolated CD73⁺, CD105⁺ and HLA-G⁺ placental cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said population of cells are isolated CD73⁺, CD105⁺, HLA-G⁺placental cells. In another embodiment, at least about 70% of cells insaid population of cells are isolated CD73⁺, CD105⁺, HLA-G⁺ placentalcells. In another embodiment, at least about 90%, 95%, or 99% of cellsin said population of cells are isolated CD73⁺, CD105⁺, HLA-G⁺ placentalcells. In a specific embodiment of the above populations, said isolatedCD73⁺, CD105⁺, HLA-G⁺ placental cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺,HLA-G⁺ placental cells are additionally CD34⁻, CD38⁻ and CD45⁻. Inanother specific embodiment, said isolated CD73⁺, CD105⁺, HLA-G⁺placental cells are additionally OCT-4′. In another specific embodiment,said isolated CD73⁺, CD105⁺, HLA-G⁺ placental cells are additionallyCD200⁺. In a more specific embodiment, said isolated CD73⁺, CD105⁺,HLA-G⁺ placental cells are additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said cell population is isolatedaway from placental cells that are not CD73⁺, CD105⁺, HLA-G⁺ placentalcells. In another specific embodiment, said cell population is isolatedaway from placental cells that do not display these markers.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are CD73⁺ and CD105⁺ andfacilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said CD73⁺, CD105⁺cells when said population is cultured under conditions that allowformation of embryoid-like bodies. In another specific embodiment, saidisolated CD73⁺, CD105⁺ placental cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are additionally CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said isolated CD73⁺, CD105⁺ placental cells areadditionally OCT-4⁺. In a more specific embodiment, said isolated CD73⁺,CD105⁺ placental cells are additionally OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, said isolated CD73⁺, CD105⁺ placentalcells are isolated away from placental cells that are not said cells. Inanother specific embodiment, said isolated CD73⁺, CD105⁺ placental cellsare isolated away from placental cells that do not display thesecharacteristics.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, isolated placental cells that are CD73⁺, CD105⁺ andfacilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said cells when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells are saidisolated CD73⁺, CD105⁺ placental cells. In another embodiment, at leastabout 70% of cells in said population of cells are said isolated CD73⁺,CD105⁺ placental cells. In another embodiment, at least about 90%, 95%or 99% of cells in said population of cells are said isolated CD73⁺,CD105⁺ placental cells. In a specific embodiment of the abovepopulations, said isolated CD73⁺, CD105⁺ placental cells areadditionally CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidisolated CD73⁺, CD105⁺ placental cells are additionally CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are additionally OCT-4⁺. In another specific embodiment,said isolated CD73⁺, CD105⁺ placental cells are additionally CD200⁺. Ina more specific embodiment, said isolated CD73⁺, CD105⁺ placental cellsare additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In anotherspecific embodiment, said cell population is isolated away fromplacental cells that are not said isolated CD73⁺, CD105⁺ placentalcells. In another specific embodiment, said cell population is isolatedaway from placental cells that do not display these markers.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are OCT-4⁺ and facilitateformation of one or more embryoid-like bodies in a population ofisolated placental cells comprising said cells when cultured underconditions that allow formation of embryoid-like bodies. In a specificembodiment, said isolated OCT-4⁺ placental cells are additionally CD73⁺and CD105⁺. In another specific embodiment, said isolated OCT-4⁺placental cells are additionally CD34⁻, CD38⁻, or CD45⁻. In anotherspecific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD200⁺. In a more specific embodiment, said isolated OCT-4⁺placental cells are additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻,and CD45⁻. In another specific embodiment, said isolated OCT-4⁺placental cells are isolated away from placental cells that are notOCT-4⁺ placental cells. In another specific embodiment, said isolatedOCT-4⁺ placental cells are isolated away from placental cells that donot display these characteristics.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, is a population of cells comprising, e.g., thatis enriched for, isolated placental cells that are OCT-4⁺ and facilitatethe formation of one or more embryoid-like bodies in a population ofisolated placental cells comprising said cells when said population iscultured under conditions that allow formation of embryoid-like bodies.In various embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said population of cells are said isolated OCT-4⁺ placentalcells. In another embodiment, at least about 70% of cells in saidpopulation of cells are said isolated OCT-4⁺ placental cells. In anotherembodiment, at least about 80%, 90%, 95% or 99% of cells in saidpopulation of cells are said isolated OCT-4⁺ placental cells. In aspecific embodiment of the above populations, said isolated OCT-4⁺placental cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said isolated OCT-4⁺ placental cells are additionally CD73⁺ and CD105⁺.In another specific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD200⁺. In a more specific embodiment, said isolated OCT-4⁺placental cells are additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻,and CD45⁻. In another specific embodiment, said cell population isisolated away from placental cells that are not said cells. In anotherspecific embodiment, said cell population is isolated away fromplacental cells that do not display these markers.

In certain other embodiments, the isolated placental cells are one ormore of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, SH4⁺, SSEA3−, SSEA4⁻, OCT-4⁺, MHC-I⁺ or ABC-p⁺. In a specificembodiment, the isolated placental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻,CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3−, SSEA4⁻, andOCT-4⁺. In another specific embodiment, the isolated placental cells areCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺, SH3⁺, and SH4⁺. Inanother specific embodiment, the isolated placental cells are CD10⁺,CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. Inanother specific embodiment, the isolated placental cells are CD10⁺,CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, HLA-1⁺, SH2⁺, SH3⁺,SH4⁺. In another specific embodiment, the isolated placental cells areOCT-4⁺ and ABC-p⁺. In another specific embodiment, the isolatedplacental cells are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. In another embodiment,the isolated placental cells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻. In aspecific embodiment, said isolated OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻placental cells are additionally CD10⁺, CD29⁺, CD34⁻, CD44⁺, CD45⁻,CD54⁺, CD90⁺, SH2⁺, SH3⁺, and SH4⁺. In another embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, and either SH3⁺ or SH4⁺. Inanother embodiment, the isolated placental cells are CD34⁻ and eitherCD10⁺, CD29⁺, CD44⁺, CD54⁺, CD90⁺, or OCT-4⁺.

In another embodiment, isolated placental cells, usable in thecompositions and methods provided herein, are isolated CD10⁺, CD34⁻,CD105⁺, and CD200⁺ placental cells. In another embodiment, a cellpopulation, usable in the compositions and methods provided herein, is apopulation of cells comprising placental cells, wherein at least about70%, at least about 80%, at least about 90%, at least about 95% or atleast about 99% of said population of cells are CD10⁺, CD34⁻, CD105⁺,CD200⁺ placental cells. In a specific embodiment of the aboveembodiments, said placental cells are additionally CD90⁺ and CD45⁻. In aspecific embodiment, said placental cell or population of placentalcells is isolated away from placental cells that are not stem cells. Inanother specific embodiment, said placental cell or population ofplacental cells is isolated away from placental cells that do notdisplay these characteristics. In another specific embodiment, saidisolated placental cell is non-maternal in origin. In another specificembodiment, at least about 90%, at least about 95%, or at least about99% of said cells in said isolated population of placental cells, arenon-maternal in origin.

In another specific embodiment of said isolated placental cells orpopulations of cells comprising the isolated placental cells, said cellsor population have been expanded, for example, passaged at least, about,or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 times, or proliferated for at least, about, or no morethan, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38 or 40 population doublings. In another specificembodiment of the isolated placental cells, or populations of cellscomprising isolated placental cells, that are disclosed herein, saidisolated placental cells are fetal in origin (that is, have the fetalgenotype).

In another embodiment, placental cells, usable in the compositions andmethods provided herein, are OCT-4⁺ and facilitate formation of one ormore embryoid-like bodies in a population of said isolated placentalcells when cultured under conditions that allow formation ofembryoid-like bodies. In a specific embodiment, said placental cells areCD73⁺ and CD105⁺. In another specific embodiment, said placental cellsare CD34⁻, CD38⁻, or CD45⁻. In another specific embodiment, saidplacental cells are CD200⁺. In a more specific embodiment, saidplacental cells are CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. Inanother specific embodiment, said placental cells are isolated away fromplacental cells that are not stem cells. In another specific embodiment,said placental cells are isolated away from placental cells that do notdisplay these characteristics.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated HLA-A,B,C⁻,CD45⁻, CD133⁻ and CD34⁻ placental cells. In another embodiment, a cellpopulation, usable in the compositions and methods provided herein, is apopulation of cells comprising isolated placental cells, wherein atleast about 70%, at least about 80%, at least about 90%, at least about95% or at least about 99% of cells in said isolated population of cellsare isolated HLA-A,B,C⁻, CD45⁻, CD133⁻ and CD34⁻ placental cells. In aspecific embodiment, said isolated placental cell or population ofisolated placental cells is isolated away from placental cells that arenot HLA-A,B,C⁻, CD45⁻, CD133⁻ and CD34⁻ placental cells. In anotherspecific embodiment, said isolated placental cells are non-maternal inorigin. In another specific embodiment, said isolated population ofplacental cells are substantially free of maternal components; e.g., atleast about 40%, 45%, 5-0%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%,98% or 99% of said cells in said isolated population of placental cellsare non-maternal in origin.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated CD10⁺, CD13⁺,CD33⁺, CD45⁻, CD117⁻ and CD133⁻ placental cells. In another embodiment,a cell population usable in the compositions and methods providedherein, is a population of cells comprising isolated placental cells,wherein at least about 70%, at least about 80%, at least about 90%, atleast about 95% or at least about 99% of cells in said population ofcells are isolated CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻placental cells. In a specific embodiment, said isolated placental cellsor population of isolated placental cells is isolated away fromplacental cells that are not said isolated placental cells. In anotherspecific embodiment, said isolated CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻and CD133⁻ placental cells are non-maternal in origin, i.e., have thefetal genotype. In another specific embodiment, at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of saidcells in said isolated population of placental cells, are non-maternalin origin. In another specific embodiment, said isolated placental cellsor population of isolated placental cells are isolated away fromplacental cells that do not display these characteristics.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated CD10⁻, CD33⁻,CD44⁺, CD45⁻, and CD117⁻ placental cells. In another embodiment, a cellpopulation, usable in the compositions and methods provided herein, is apopulation of cells comprising, e.g., enriched for, isolated placentalcells, wherein at least about 70%, at least about 80%, at least about90%, at least about 95% or at least about 99% of cells in saidpopulation of cells are isolated CD10⁻, CD33⁻, CD44⁺, CD45⁻, and CD117⁻placental cells. In a specific embodiment, said isolated placental cellor population of isolated placental cells is isolated away fromplacental cells that are not said cells. In another specific embodiment,said isolated placental cells are non-maternal in origin. In anotherspecific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cellpopulation are non-maternal in origin. In another specific embodiment,said isolated placental cell or population of isolated placental cellsis isolated away from placental cells that do not display these markers.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated CD10⁻, CD13⁻,CD33⁻, CD45⁻, and CD117⁻ placental cells. In another embodiment, a cellpopulation useful, usable in the compositions and methods providedherein, is a population of cells comprising, e.g., enriched for,isolated CD10⁻, CD13⁻, CD33⁻, CD45⁻, and CD117⁻ placental cells, whereinat least about 70%, at least about 80%, at least about 90%, at leastabout 95% or at least about 99% of cells in said population are CD10⁻,CD13⁻, CD33⁻, CD45⁻, and CD117⁻ placental cells. In a specificembodiment, said isolated placental cells or population of isolatedplacental cells are isolated away from placental cells that are not saidcells. In another specific embodiment, said isolated placental cells arenon-maternal in origin. In another specific embodiment, at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99%of said cells in said cell population are non-maternal in origin. Inanother specific embodiment, said isolated placental cells or populationof isolated placental cells is isolated away from placental cells thatdo not display these characteristics.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are HLA A,B,C⁺, CD45⁻, CD34⁻,and CD133⁻, and are additionally CD10⁺, CD13⁺, CD38⁺, CD44⁺, CD90⁺,CD105⁺, CD200⁺ and/or HLA-G⁺, and/or negative for CD117. In anotherembodiment, a cell population, usable in the compositions and methodsprovided herein, is a population of cells comprising isolated placentalcells, wherein at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% of the cells insaid population are isolated placental cells that are HLA A,B,C⁻, CD45⁻,CD34⁻, CD133⁻, and that are additionally positive for CD10, CD13, CD38,CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117. In aspecific embodiment, said isolated placental cells or population ofisolated placental cells are isolated away from placental cells that arenot said cells. In another specific embodiment, said isolated placentalcells are non-maternal in origin. In another specific embodiment, atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%,98% or 99% of said cells in said cell population are non-maternal inorigin. In another specific embodiment, said isolated placental cells orpopulation of isolated placental cells are isolated away from placentalcells that do not display these markers.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated placental cellsthat are CD200⁺ and CD10⁺, as determined by antibody binding, andCD117⁺, as determined by both antibody binding and RT-PCR. In anotherembodiment, the isolated placental cells, usable in the compositions andmethods provided herein, are isolated placental cells, e.g., placentalstem cells or placental multipotent cells, that are CD10⁺, CD29⁻, CD54⁺,CD200⁺, HLA-G⁺, HLA class I⁻ and β-2-microglobulin. In anotherembodiment, isolated placental cells, usable in the compositions andmethods provided herein, are placental cells wherein the expression ofat least one cellular marker is at least two-fold higher than for amesenchymal stem cell (e.g., a bone marrow-derived mesenchymal stemcell). In another specific embodiment, said isolated placental cells arenon-maternal in origin. In another specific embodiment, at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99%of said cells in said cell population are non-maternal in origin.

In another embodiment, the isolated placental cells, usable in thecompositions and methods provided herein, are isolated placental cells,e.g., placental stem cells or placental multipotent cells, that are oneor more of CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻,CD62-P⁻, CD80⁻, CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺,CD144/VE-cadherin^(low), CD184/CXCR4⁻, β2-microglobulin^(low), MHC-II⁻,HLA-G^(low), and/or PDL1^(low). In a specific embodiment, the isolatedplacental cells are at least CD29⁺ and CD54⁺. In another specificembodiment, the isolated placental cells are at least CD44⁺ and CD106⁺.In another specific embodiment, the isolated placental cells are atleast CD29⁺.

In another embodiment, a cell population, usable in the compositions andmethods provided herein, comprises isolated placental cells, and atleast 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said cellpopulation are isolated placental cells that are one or more of CD10⁺,CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻, CD62-P⁻, CD80⁻,CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(low),CD184/CXCR4⁻, β2-microglobulin^(low), MHC-I^(low), MHC-II⁻, HLA-G^(low),and/or PDL1^(low). In a more specific embodiment, at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% of cells in said cell population areCD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻, CD62-P⁻,CD80⁻, CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺,CD144/VE-cadherin^(low), CD184/CXCR4⁻, β2-microglobulin^(low),MHC-I^(low), MHC-II⁻, HLA-G^(low), and PDL1^(low).

In certain embodiments of isolated placental cells, said isolatedplacental cells do not differentiate during culturing in growth medium,i.e., medium formulated to promote proliferation, e.g., duringproliferation in growth medium. In another specific embodiment, saidisolated placental cells do not require a feeder layer in order toproliferate. In another specific embodiment, said isolated placentalcells do not differentiate in culture in the absence of a feeder layer,solely because of the lack of a feeder cell layer.

In another embodiment, cells, usable in the compositions and methodsprovided herein, are isolated placental cells, wherein a plurality ofsaid isolated placental cells are positive for aldehyde dehydrogenase(ALDH), as assessed by an aldehyde dehydrogenase activity assay. Suchassays are known in the art (see, e.g., Bostian and Betts, Biochem. J.,173, 787, (1978)). In a specific embodiment, said ALDH assay usesALDEFLUOR® (Aldagen, Inc., Ashland, Oreg.) as a marker of aldehydedehydrogenase activity. In a specific embodiment, said plurality isbetween about 3% and about 25% of cells in said population of cells. Inanother embodiment, provided herein is a population of isolatedumbilical cord cells, e.g., multipotent isolated umbilical cord cells,wherein a plurality of said isolated umbilical cord cells are positivefor aldehyde dehydrogenase, as assessed by an aldehyde dehydrogenaseactivity assay that uses ALDEFLUOR® as an indicator of aldehydedehydrogenase activity. In a specific embodiment, said plurality isbetween about 3% and about 25% of cells in said population of cells. Inanother embodiment, said population of isolated placental cells orisolated umbilical cord cells shows at least three-fold, or at leastfive-fold, higher ALDH activity than a population of bone marrow-derivedmesenchymal stem cells having about the same number of cells andcultured under the same conditions.

In another specific embodiment of said isolated placental cells orpopulations of cells comprising the isolated placental cells, said cellsor population have been expanded, for example, passaged at least, about,or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 times, or proliferated for at least, about, or no morethan, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38 or 40 population doublings. In another specificembodiment of the isolated placental cells, or populations of cellscomprising isolated placental cells, that are disclosed herein, saidisolated placental cells are fetal in origin (that is, have the fetalgenotype).

In a specific embodiment of any of the above isolated placental cells orcell populations of isolated placental cells, the karyotype of thecells, or at least about 95% or about 99% of the cells in saidpopulation, is normal. In another specific embodiment of any of theabove placental cells or cell populations, the cells, or cells in thepopulation of cells, are non-maternal in origin.

Isolated placental cells, or populations of isolated placental cells,bearing any of the above combinations of markers, can be combined in anyratio. Any two or more of the above isolated placental cell populationscan be combined to form an isolated placental cell population. Forexample, an population of isolated placental cells can comprise a firstpopulation of isolated placental cells defined by one of the markercombinations described above, and a second population of isolatedplacental cells defined by another of the marker combinations describedabove, wherein said first and second populations are combined in a ratioof about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40:60,50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about99:1. In like fashion, any three, four, five or more of theabove-described isolated placental cells or isolated placental cellspopulations can be combined.

Isolated placental cells, usable in the compositions and methodsprovided herein, can be obtained, e.g., by disruption of placentaltissue, with or without enzymatic digestion (see Section 5.4.3) orperfusion (see Section 5.4.4). For example, populations of isolatedplacental cells can be produced according to a method comprisingperfusing a mammalian placenta that has been drained of cord blood andperfused to remove residual blood; perfusing said placenta with aperfusion solution; and collecting said perfusion solution, wherein saidperfusion solution after perfusion comprises a population of placentalcells that comprises isolated placental cells; and isolating a pluralityof said isolated placental cells from said population of cells. In aspecific embodiment, the perfusion solution is passed through both theumbilical vein and umbilical arteries and collected after it exudes fromthe placenta. In another specific embodiment, the perfusion solution ispassed through the umbilical vein and collected from the umbilicalarteries, or passed through the umbilical arteries and collected fromthe umbilical vein.

In various embodiments, the isolated placental cells, contained within apopulation of cells obtained from perfusion of a placenta, are at least50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said populationof placental cells. In another specific embodiment, the isolatedplacental cells collected by perfusion comprise fetal and maternalcells. In another specific embodiment, the isolated placental cellscollected by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% orat least 99.5% fetal cells.

In another specific embodiment, provided herein is a compositioncomprising a population of the isolated placental cells, as describedherein, collected by perfusion, wherein said composition comprises atleast a portion of the perfusion solution used to collect the isolatedplacental cells.

Isolated populations of the isolated placental cells described hereincan be produced by digesting placental tissue with a tissue-disruptingenzyme to obtain a population of placental cells comprising the cells,and isolating, or substantially isolating, a plurality of the placentalcells from the remainder of said placental cells. The whole, or any partof, the placenta can be digested to obtain the isolated placental cellsdescribed herein. In specific embodiments, for example, said placentaltissue can be a whole placenta, an amniotic membrane, chorion, acombination of amnion and chorion, or a combination of any of theforegoing. In other specific embodiment, the tissue-disrupting enzyme istrypsin or collagenase. In various embodiments, the isolated placentalcells, contained within a population of cells obtained from digesting aplacenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least99.5% of said population of placental cells.

Gene profiling confirms that isolated placental cells, and populationsof isolated placental cells, are distinguishable from other cells, e.g.,mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells. The isolated placental cells described herein can bedistinguished from, e.g., mesenchymal stem cells on the basis of theexpression of one or more genes, the expression of which issignificantly higher in the isolated placental cells, or in certainisolated umbilical cord stem cells, in comparison to bone marrow-derivedmesenchymal stem cells. In particular, the isolated placental cells,usable in the compositions and methods provided herein, can bedistinguished from mesenchymal stem cells on the basis of the expressionof one or more genes, the expression of which is significantly higher(that is, at least twofold higher) in the isolated placental cells thanin an equivalent number of bone marrow-derived mesenchymal stem cells,wherein the one or more genes are ACTG2, ADARB1, AMIGO2, ARTS-1,B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2,ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3,IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3,NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5,SLC12A8, TCF21, TGFB2, VTN, ZC3H12A, or a combination of any of theforegoing, when the cells are grown under equivalent conditions. See,e.g., U.S. Patent Application Publication No. 2007/0275362, thedisclosure of which is incorporated herein by reference in its entirety.In a more specific embodiment, said isolated placental cells expresssaid one or more genes when cultured for from about 3 to about 35population doublings in a medium comprising DMEM-LG (Gibco); 2% fetalcalf serum (Hyclone Labs); 1× insulin-transferrin-selenium (ITS); 1×linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹ M dexamethasone(Sigma); 10⁻⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (R&D Systems). In a specific embodiment, the isolatedplacental cell-specific or isolated umbilical cord cell-specific gene isCD200.

Specific sequences for these genes can be found in GenBank at accessionnos. NM_001615 (ACTG2), BC065545 (ADARB1), (NM_181847 (AMIGO2), AY358590(ARTS-1), BC074884 (B4GALT6), BC008396 (BCHE), BC020196 (C11orf9),BC031103 (CD200), NM_001845 (COL4A1), NM_001846 (COL4A2), BC052289(CPA4), BC094758 (DMD), AF293359 (DSC3), NM_001943 (DSG2), AF338241(ELOVL2), AY336105 (F2RL1), NM_018215 (FLJ10781), AY416799 (GATA6),BC075798 (GPR126), NM_016235 (GPRC5B), AF340038 (ICAM1), BC000844(IER3), BC066339 (IGFBP7), BC013142 (IL1A), BT019749 (IL6), BC007461(IL18), (BC072017) KRT18, BC075839 (KRT8), BC060825 (LIPG), BC065240(LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3), NM_014840(NUAK1), AB006755 (PCDH7), NM_014476 (PDLIM3), BC126199 (PKP-2),BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201(ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21), BC096235(TGFB2), BC005046 (VTN), and BC005001 (ZC3H12A) as of March 2008.

In a more specific embodiment, said isolated placental cells expresseach of ACTG2, ADARB1, AMIGO2, ARTS-I, B4GALT6, BCHE, C11orf9, CD200,COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6,GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18,KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1,SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12Aat a detectably higher level than an equivalent number of bonemarrow-derived mesenchymal stem cells, when the cells are grown underequivalent conditions.

In more specific embodiments, placental cell populations can be selectedby selecting placental cells that express one or more genes at adetectably higher level than a bone marrow-derived mesenchymal stemcell, wherein said one or more genes are selected from the groupconsisting of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9,CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781,GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18,KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3,PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN,and ZC3H12A, and wherein said bone marrow derived stem cell hasundergone a number of passages in culture equivalent to the number ofpassages said placental cell has undergone. In a more specificembodiment, said selecting comprises selecting cells that express ACTG2,ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2,CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B,HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP,MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9,ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A at adetectably higher level than a bone marrow-derived mesenchymal stemcell.

Expression of the above-referenced genes can be assessed by standardtechniques. For example, probes based on the sequence of the gene(s) canbe individually selected and constructed by conventional techniques.Expression of the genes can be assessed, e.g., on a microarraycomprising probes to one or more of the genes, e.g., an AffymetrixGENECHIP® Human Genome U133A 2.0 array, or an Affymetrix GENECHIP® HumanGenome U133 Plus 2.0 (Santa Clara, Calif.). Expression of these genescan be assessed even if the sequence for a particular GenBank accessionnumber is amended because probes specific for the amended sequence canreadily be generated using well-known standard techniques.

The level of expression of these genes can be used to confirm theidentity of a population of isolated placental cells, to identify apopulation of cells as comprising at least a plurality of isolatedplacental cells, or the like. Populations of isolated placental cells,the identity of which is confirmed, can be clonal, e.g., populations ofisolated placental cells expanded from a single isolated placental cell,or a mixed population of stem cells, e.g., a population of cellscomprising solely isolated placental cells that are expanded frommultiple isolated placental cells, or a population of cells comprisingisolated placental cells, as described herein, and at least one othertype of cell.

The level of expression of these genes can be used to select populationsof isolated placental cells. For example, a population of cells, e.g.,clonally-expanded cells, may be selected if the expression of one ormore of the genes listed above is significantly higher in a sample fromthe population of cells than in an equivalent population of mesenchymalstem cells. Such selecting can be of a population from a plurality ofisolated placental cell populations, from a plurality of cellpopulations, the identity of which is not known, etc.

Isolated placental cells can be selected on the basis of the level ofexpression of one or more such genes as compared to the level ofexpression in said one or more genes in, e.g., a mesenchymal stem cellcontrol, for example, the level of expression in said one or more genesin an equivalent number of bone marrow-derived mesenchymal stem cells.In one embodiment, the level of expression of said one or more genes ina sample comprising an equivalent number of mesenchymal stem cells isused as a control. In another embodiment, the control, for isolatedplacental cells tested under certain conditions, is a numeric valuerepresenting the level of expression of said one or more genes inmesenchymal stem cells under said conditions.

The isolated placental cells described herein display the abovecharacteristics (e.g., combinations of cell surface markers and/or geneexpression profiles) in primary culture, or during proliferation inmedium comprising, e.g., DMEM-LG (Gibco), 2% fetal calf serum (FCS)(Hyclone Laboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹ M dexamethasone(Sigma), 10⁻⁴ M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 Ustreptomycin.

The isolated populations of placental cells described above, andpopulations of isolated placental cells generally, can comprise about,at least, or no more than, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or more of theisolated placental cells. Populations of isolated placental cells usablein the compositions and methods provided herein comprise at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% viable isolatedplacental cells, as determined by, e.g., trypan blue exclusion.

5.3.3 Growth in Culture

The growth of the isolated placental cells described herein, as for anymammalian cell, depends in part upon the particular medium selected forgrowth. Under optimum conditions, isolated placental cells typicallydouble in number in 3-5 days. During culture, the isolated placentalcells described herein adhere to a substrate in culture, e.g. thesurface of a tissue culture container (e.g., tissue culture dishplastic, fibronectin-coated plastic, and the like) and form a monolayer.

Populations of isolated placental cells described herein, when culturedunder appropriate conditions, form embryoid-like bodies, that is,three-dimensional clusters of cells grow atop the adherent stem celllayer. Cells within the embryoid-like bodies express markers associatedwith very early stem cells, e.g., OCT-4, Nanog, SSEA3 and SSEA4. Cellswithin the embryoid-like bodies are typically not adherent to theculture substrate, as are the isolated placental cells described herein,but remain attached to the adherent cells during culture. Embryoid-likebody cells are dependent upon the adherent isolated placental cells forviability, as embryoid-like bodies do not form in the absence of theadherent isolated placental cells. The adherent isolated placental cellsthus facilitate the growth of one or more embryoid-like bodies in apopulation of placental cells that comprise the adherent isolatedplacental cells. Without wishing to be bound by theory, the cells of theembryoid-like bodies are thought to grow on the adherent isolatedplacental cells much as embryonic stem cells grow on a feeder layer ofcells. Mesenchymal stem cells, e.g., bone marrow-derived mesenchymalstem cells, do not develop embryoid-like bodies in culture.

5.3.4 Hematopoietic Placental Stem Cells

In certain embodiments, the isolated placental cells are CD34⁺ placentalcells, e.g., hematopoietic placental cells. Such CD34⁺ cells are not,however, encompassed by the term “multipotent” as used herein. Suchcells are obtainable from placental tissue, e.g., from a placenta thathas been drained of cord blood and perfused to remove residual blood. Incertain embodiments, the CD34⁺ isolated placental cells are CD38⁺. Incertain embodiments, the CD34⁺ isolated placental cells are CD38⁻. Incertain other embodiments, the CD34⁺ isolated placental cells are CD45⁺.In a specific embodiment, the isolated placental cells are CD34⁺, CD38⁻and CD45⁺.

5.3.5 Placental Perfusate Cells

In certain embodiments, the cells of the compositions provided herein,formulated by the methods provided herein, are cells obtained fromplacental perfusate. As used herein, “cells obtained from placentalperfusate” includes total nucleated cells obtained from, e.g., isolatedfrom, placental perfusate, a subset of nucleated cells obtained fromplacental perfusate, or cells cultured or proliferated from cellsobtained directly from placental perfusate. Placental perfusate may beobtained from a placenta that has been drained of cord blood andperfused to remove residual blood, prior to perfusion to obtainplacental cells. Placental perfusate may be obtained from a placentathat has been drained of cord blood but not perfused to remove residualblood. Placental perfusate may be obtained from a placenta that hasneither been drained of cord blood nor perfused to remove residualblood. In the latter two embodiments, the placental cells, e.g.,nucleated cells from placental perfusate, for example, total nucleatedcells from placental perfusate, comprise nucleated cells from placentalblood and/or cord blood. Methods for obtaining placental perfusate, andcells from placental perfusate, are described in Section 5.4.4, below.

5.4 Methods of Obtaining Isolated Placental Cells

5.4.1 Stem Cell Collection Composition

Further provided herein are methods of collecting and isolatingplacental cells e.g., the isolated placental cells described in Section5.2, above. Generally, such cells are obtained from a mammalian placentausing a physiologically-acceptable solution, e.g., a stem cellcollection composition. A cell collection composition is described indetail in related U.S. Patent Application Publication No. 2007/0190042,entitled “Improved Medium for Collecting Placental Stem Cells andPreserving Organs.”

The cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of cells, e.g., the isolated placental cells described herein,for example, a saline solution (e.g., phosphate-buffered saline, Kreb'ssolution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.),a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.

The cell collection composition can comprise one or more components thattend to preserve isolated placental cells, that is, prevent the isolatedplacental cells from dying, or delay the death of the isolated placentalcells, reduce the number of isolated placental cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The cell collection composition can also comprise one or more of thefollowing compounds: adenosine (about 1 mM to about 50 mM); D-glucose(about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50mM); a macromolecule of molecular weight greater than 20,000 daltons, inone embodiment, present in an amount sufficient to maintain endothelialintegrity and cellular viability (e.g., a synthetic or naturallyoccurring colloid, a polysaccharide such as dextran or a polyethyleneglycol present at about 25 g/l to about 100 g/1, or about 40 g/l toabout 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylatedhydroxytoluene, glutathione, vitamin C or vitamin E present at about 25μM to about 100 μM); a reducing agent (e.g., N-acetylcysteine present atabout 0.1 mM to about 5 mM); an agent that prevents calcium entry intocells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.4.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental stem cells can beused, in light of the medical history, for personalized medicine for theinfant associated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of isolated placental cells, the umbilical cord bloodand placental blood are removed. In certain embodiments, after delivery,the cord blood in the placenta is recovered. The placenta can besubjected to a conventional cord blood recovery process. Typically aneedle or cannula is used, with the aid of gravity, to exsanguinate theplacenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al.,U.S. Pat. No. 5,415,665). The needle or cannula is usually placed in theumbilical vein and the placenta can be gently massaged to aid indraining cord blood from the placenta. Such cord blood recovery may beperformed commercially, e.g., LifeBank USA, Cedar Knolls, N.J., ViaCord,Cord Blood Registry and Cryocell. Preferably, the placenta is gravitydrained without further manipulation so as to minimize tissue disruptionduring cord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. Pat. No. 7,147,626. Preferably, the placenta is deliveredto the laboratory four to twenty-four hours following delivery. Incertain embodiments, the proximal umbilical cord is clamped, preferablywithin 4-5 cm (centimeter) of the insertion into the placental discprior to cord blood recovery. In other embodiments, the proximalumbilical cord is clamped after cord blood recovery but prior to furtherprocessing of the placenta.

The placenta, prior to cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5 to25° C. (centigrade). The placenta may be stored for a period of for aperiod of four to twenty-four hours, up to forty-eight hours, or longerthan forty eight hours, prior to perfusing the placenta to remove anyresidual cord blood. In one embodiment, the placenta is harvested frombetween about zero hours to about two hours post-expulsion. The placentais preferably stored in an anticoagulant solution at a temperature of 5to 25° C. (centigrade). Suitable anticoagulant solutions are well knownin the art. For example, a solution of heparin or warfarin sodium can beused. In a preferred embodiment, the anticoagulant solution comprises asolution of heparin (e.g., 1% w/w in 1:1000 solution). The exsanguinatedplacenta is preferably stored for no more than 36 hours before placentalcells are collected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain isolated placental cells.

5.4.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, stem cells are collected from a mammalian placenta byphysical disruption of part of all of the organ. For example, theplacenta, or a portion thereof, may be, e.g., crushed, sheared, minced,diced, chopped, macerated or the like. The tissue can then be culturedto obtain a population of isolated placental cells. Typically, theplacental tissue is disrupted using, e.g., in, a placental cellcollection composition (see Section 5.2 and below).

The placenta can be dissected into components prior to physicaldisruption and/or enzymatic digestion and stem cell recovery. Placentalstem cells can be obtained from all or a portion of the amnioticmembrane, chorion, umbilical cord, placental cotyledons, or anycombination thereof, including from a whole placenta. Preferably,isolated placental cells are obtained from placental tissue comprisingamnion and chorion. Typically, isolated placental cells can be obtainedby disruption of a small block of placental tissue, e.g., a block ofplacental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 orabout 1000 cubic millimeters in volume. Any method of physicaldisruption can be used, provided that the method of disruption leaves aplurality, more preferably a majority, and more preferably at least 60%,70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable, asdetermined by, e.g., trypan blue exclusion.

Stem cells can generally be collected from a placenta, or portionthereof, at any time within about the first three days post-expulsion,but preferably between about 8 hours and about 18 hours post-expulsion.

In a specific embodiment, the disrupted tissue is cultured in tissueculture medium suitable for the proliferation of isolated placentalcells (see, e.g., Section 5.5, below, describing the culture of isolatedplacental cells).

In another specific embodiment, isolated placental are collected byphysical disruption of placental tissue, wherein the physical disruptionincludes enzymatic digestion, which can be accomplished by use of one ormore tissue-digesting enzymes. The placenta, or a portion thereof, mayalso be physically disrupted and digested with one or more enzymes, andthe resulting material then immersed in, or mixed into, a cellcollection composition.

A preferred cell collection composition comprises one or moretissue-disruptive enzyme(s). Enzymatic digestion preferably uses acombination of enzymes, e.g., a combination of a matrix metalloproteaseand a neutral protease, for example, a combination of collagenase anddispase. In one embodiment, enzymatic digestion of placental tissue usesa combination of a matrix metalloprotease, a neutral protease, and amucolytic enzyme for digestion of hyaluronic acid, such as a combinationof collagenase, dispase, and hyaluronidase or a combination of LIBERASE(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Otherenzymes that can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental cells, e.g., placental stemcells and placental multipotent cells. For example, in one embodiment, aplacenta, or part thereof, is digested first with an appropriate amountof collagenase I at about 1 to about 2 mg/ml for, e.g., 30 minutes,followed by digestion with trypsin, at a concentration of about 0.25%,for, e.g., 10 minutes, at 37° C. Serine proteases are preferably usedconsecutively following use of other enzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the placental cell collection composition, or to asolution in which the tissue is disrupted and/or digested prior toisolation of the placental cells with the placental cell collectioncomposition.

Following digestion, the digestate is washed, for example three times,with culture medium, and the washed cells are seeded into cultureflasks. The cells are then isolated by differential adherence, andcharacterized for, e.g., viability, cell surface markers,differentiation, and the like.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental cells collected will comprise a mix of placental cells, e.g.,placental stem cells or placental multipotent cells, derived from bothfetal and maternal sources. Where a portion of the placenta thatcomprises no, or a negligible number of, maternal cells (for example,amnion), the placental cells collected will comprise almost exclusivelyfetal placental cells, e.g., fetal placental stem cells or fetalplacental multipotent cells.

Placental cells can be isolated from disrupted tissue by differentialtrypsinization (see Section 5.4.5, below) followed by culture in one ormore new culture containers in fresh proliferation medium, optionallyfollowed by a second differential trypsinization step.

5.4.4 Placental Perfusion

Placental cells, e.g., placental stem cells or placental multipotentcells, can also be obtained by perfusion of the mammalian placenta.Methods of perfusing mammalian placenta to obtain placental cells aredisclosed, e.g., in Hariri, U.S. Pat. Nos. 7,045,148 and 7,255,729, andin related U.S. Patent Application Publication No. 2007/0190042, each ofwhich is incorporated herein in its entirety.

Placental cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a cell collection composition as aperfusion solution. In one embodiment, a mammalian placenta is perfusedby passage of perfusion solution through either or both of the umbilicalartery and umbilical vein. The flow of perfusion solution through theplacenta may be accomplished using, e.g., gravity flow into theplacenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid through the placentalvasculature and surrounding tissue. The placenta can also be perfused bypassage of a perfusion fluid into the umbilical vein and collection fromthe umbilical arteries, or passage of a perfusion fluid into theumbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilicalvein are connected simultaneously, e.g., to a pipette that is connectedvia a flexible connector to a reservoir of the perfusion solution. Theperfusion solution is passed into the umbilical vein and artery. Theperfusion solution exudes from and/or passes through the walls of theblood vessels into the surrounding tissues of the placenta, and iscollected in a suitable open vessel from the surface of the placentathat was attached to the uterus of the mother during gestation. Theperfusion solution may also be introduced through the umbilical cordopening and allowed to flow or percolate out of openings in the wall ofthe placenta which interfaced with the maternal uterine wall. Placentalcells that are collected by this method, which can be referred to as a“pan” method, are typically a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is passed through theumbilical veins and collected from the umbilical artery, or is passedthrough the umbilical artery and collected from the umbilical veins.Placental cells collected by this method, which can be referred to as a“closed circuit” method, are typically almost exclusively fetal.

It will be appreciated that perfusion using the pan method, that is,whereby perfusate is collected after it has exuded from the maternalside of the placenta, results in a mix of fetal and maternal cells. As aresult, the cells collected by this method comprise a mixed populationof placental cells e.g., placental stem cells or placental multipotentcells, of both fetal and maternal origin. In contrast, perfusion solelythrough the placental vasculature in the closed circuit method, wherebyperfusion fluid is passed through one or two placental vessels and iscollected solely through the remaining vessel(s), results in thecollection of a population of placental cells almost exclusively offetal origin.

The closed circuit perfusion method can, in one embodiment, be performedas follows. A post-partum placenta is obtained within about 48 hoursafter birth. The umbilical cord is clamped and cut above the clamp. Theumbilical cord can be discarded, or can processed to recover, e.g.,umbilical cord stem cells, and/or to process the umbilical cord membranefor the production of a biomaterial. The amniotic membrane can beretained during perfusion, or can be separated from the chorion, e.g.,using blunt dissection with the fingers. If the amniotic membrane isseparated from the chorion prior to perfusion, it can be, e.g.,discarded, or processed, e.g., to obtain placental cells by enzymaticdigestion, or to produce, e.g., an amniotic membrane biomaterial, e.g.,the biomaterial described in U.S. Application Publication No.2004/0048796. After cleaning the placenta of all visible blood clots andresidual blood, e.g., using sterile gauze, the umbilical cord vesselsare exposed, e.g., by partially cutting the umbilical cord membrane toexpose a cross-section of the cord. The vessels are identified, andopened, e.g., by advancing a closed alligator clamp through the cut endof each vessel. The apparatus, e.g., plastic tubing connected to aperfusion device or peristaltic pump, is then inserted into each of theplacental arteries. The pump can be any pump suitable for the purpose,e.g., a peristaltic pump. Plastic tubing, connected to a sterilecollection reservoir, e.g., a blood bag such as a 250 mL collection bag,is then inserted into the placental vein. Alternatively, the tubingconnected to the pump is inserted into the placental vein, and tubes toa collection reservoir(s) are inserted into one or both of the placentalarteries. The placenta is then perfused with a volume of perfusionsolution, e.g., about 750 ml of perfusion solution. Cells in theperfusate are then collected, e.g., by centrifugation.

In another embodiment, perfusion, e.g., to collect placental perfusatecells, is performed as follows. Placenta(e) containing placental bloodare perfused through only the placental vasculature by pumping sterile0.9% NaCl (e.g., about 750 mL) using, e.g., a peristaltic pump, and theresulting perfusate is collected in a collection bag. Cells from theperfusate are collected by centrifugation, e.g., at about 420 g,followed by removal of excess supernatant (NaCl, plasma, anticoagulant).Hetastarch is then added to the perfusate cells to obtain a 30%dilution. The perfusate cells are then placed into a plasma extractor,e.g., for about an hour, to separate erythrocytes. Resulting plasma andnucleated cells are separated from the collection bag, and placed againin a plasma extractor. Remaining cells are resuspended in 5% human serumalbumin in a final volume of about 20 mL. Premixed DMSO/PLASMALYTE A®(1:1 v/v) is added to obtain a volume of about 24 mL. The resultingcells are cryopreserved. In specific embodiments of this method, theplacenta from which the perfusate is obtained is drained of cord blood,but not perfused, prior to perfusion to collect placental cells. Inanother specific embodiment, the placenta from which the perfusate isobtained is drained of cord blood, and is perfused to remove residualblood, prior to perfusion to collect placental cells.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The volume of perfusion liquid used to collect placental cells may varydepending upon the number of cells to be collected, the size of theplacenta, the number of collections to be made from a single placenta,etc. In various embodiments, the volume of perfusion liquid may be from50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL,250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically,the placenta is perfused with 700-800 mL of perfusion liquid followingexsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe cell collection composition, or a standard perfusion solution (e.g.,a normal saline solution such as phosphate buffered saline (“PBS”)) withor without an anticoagulant (e.g., heparin, warfarin sodium, coumarin,bishydroxycoumarin), and/or with or without an antimicrobial agent(e.g., β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin(e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B(e.g., at 0.5 μg/ml). In one embodiment, an isolated placenta ismaintained or cultured for a period of time without collecting theperfusate, such that the placenta is maintained or cultured for 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours, or 2 or 3 or more days before perfusion and collectionof perfusate. The perfused placenta can be maintained for one or moreadditional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused asecond time with, e.g., 700-800 mL perfusion fluid. The placenta can beperfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3,4, 5 or 6 hours. In a preferred embodiment, perfusion of the placentaand collection of perfusion solution, e.g., cell collection composition,is repeated until the number of recovered nucleated cells falls below100 cells/ml. The perfusates at different time points can be furtherprocessed individually to recover time-dependent populations of cells,e.g., stem cells. Perfusates from different time points can also bepooled. In a preferred embodiment, stem cells are collected at a time ortimes between about 8 hours and about 18 hours post-expulsion.

Perfusion preferably results in the collection of significantly moreplacental cells than the number obtainable from a mammalian placenta notperfused with said solution, and not otherwise treated to obtainplacental cells (e.g., by tissue disruption, e.g., enzymatic digestion).In this context, “significantly more” means at least 10% more. Perfusionyields significantly more placental stem cells than, e.g., the number ofplacental cells obtainable from culture medium in which a placenta, orportion thereof, has been cultured.

Placental cells can be isolated from placenta by perfusion with asolution comprising one or more proteases or other tissue-disruptiveenzymes. In a specific embodiment, a placenta or portion thereof (e.g.,amniotic membrane, amnion and chorion, placental lobule or cotyledon,umbilical cord, or combination of any of the foregoing) is brought to25-37° C., and is incubated with one or more tissue-disruptive enzymesin 200 mL of a culture medium for 30 minutes. Cells from the perfusateare collected, brought to 4° C., and washed with a cold inhibitor mixcomprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol.The placental cells are washed after several minutes with a cold (e.g.,4° C.) stem cell collection composition.

5.4.5 Isolation, Sorting, and Characterization of Placental Stem Cells

The isolated placental cells, e.g., the cells described in Section 5.3,above, whether obtained by perfusion or by physical disruption, e.g., byenzymatic digestion, can initially be purified from (i.e., be isolatedfrom) other cells by Ficoll gradient centrifugation. Such centrifugationcan follow any standard protocol for centrifugation speed, etc. In oneembodiment, for example, cells collected from the placenta are recoveredfrom perfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,N.Y.). The total mononuclear cell fraction can be isolated, e.g., usingLymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure.

Placental cells obtained by perfusion or digestion can, for example, befurther, or initially, isolated by differential trypsinization using,e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because the isolated placentalcells typically detach from plastic surfaces within about five minuteswhereas other adherent populations typically require more than 20-30minutes incubation. The detached placental cells can be harvestedfollowing trypsinization and trypsin neutralization, using, e.g.,Trypsin Neutralizing Solution (TNS, Cambrex). In one embodiment ofisolation of adherent cells, aliquots of, for example, about 5−10×10⁶cells are placed in each of several T-75 flasks, preferablyfibronectin-coated T75 flasks. In such an embodiment, the cells can becultured with commercially available Mesenchymal Stem Cell Growth Medium(MSCGM) (Cambrex), and placed in a tissue culture incubator (37° C., 5%CO₂). After 10 to 15 days, non-adherent cells are removed from theflasks by washing with PBS. The PBS is then replaced by MSCGM. Flasksare preferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34⁻, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34; if so, the cell is CD34⁺. Likewise, if a cellproduces enough OCT-4 RNA to be detectable by RT-PCR, or significantlymore OCT-4 RNA than an adult cell, the cell is OCT-4⁺ Antibodies to cellsurface markers (e.g., CD markers such as CD34) and the sequence of stemcell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laserexcitation of fluorescent moieties in the individual particles resultsin a small electrical charge allowing electromagnetic separation ofpositive and negative particles from a mixture. In one embodiment, cellsurface marker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, cells from placenta, e.g., placental stem cellsand placental multipotent cells, are sorted on the basis of expressionof the markers CD34⁻, CD38, CD44, CD45, CD73, CD105, OCT-4 and/or HLA-G.This can be accomplished in connection with procedures to select stemcells on the basis of their adherence properties in culture. Forexample, an adherence selection stem can be accomplished before or aftersorting on the basis of marker expression. In one embodiment, forexample, cells are sorted first on the basis of their expression ofCD34; CD34⁻ cells are retained, and cells that are CD200⁺ HLA-G⁺, areseparated from all other CD34⁻ cells. In another embodiment, cells fromplacenta are based on their expression of markers CD200 and/or HLA-G;for example, cells displaying either of these markers are isolated forfurther use. Cells that express, e.g., CD200 and/or HLA-G can, in aspecific embodiment, be further sorted based on their expression of CD73and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, orlack of expression of CD34⁻, CD38 or CD45. For example, in oneembodiment, placental cells are sorted by expression, or lack thereof,of CD200, HLA-G, CD73, CD105, CD34⁻, CD38 and CD45, and placental cellsthat are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ areisolated from other placental cells for further use.

With respect to antibody-mediated detection and sorting of placentalcells, e.g., placental stem cells or placental multipotent cells, anyantibody, specific for a particular marker, can be used, in combinationwith any fluorophore or other label suitable for the detection andsorting of cells (e.g., fluorescence-activated cell sorting).Antibody/fluorophore combinations to specific markers include, but arenot limited to, fluorescein isothiocyanate (FITC) conjugated monoclonalantibodies against HLA-G (available from Serotec, Raleigh, N.C.), CD10(available from BD Immunocytometry Systems, San Jose, Calif.), CD44(available from BD Biosciences Pharmingen, San Jose, Calif.), and CD105(available from R&D Systems Inc., Minneapolis, Minn.); phycoerythrin(PE) conjugated monoclonal antibodies against CD44, CD200, CD117, andCD13 (BD Biosciences Pharmingen); phycoerythrin-Cy7 (PE Cy7) conjugatedmonoclonal antibodies against CD33 and CD10 (BD Biosciences Pharmingen);allophycocyanin (APC) conjugated streptavidin and monoclonal antibodiesagainst CD38 (BD Biosciences Pharmingen); and biotinylated CD90 (BDBiosciences Pharmingen). Other antibodies that can be used include, butare not limited to, CD133-APC (Miltenyi), KDR-Biotin (CD309, Abeam),Cytokeratin K-FITC (Sigma or Dako), HLA ABC-FITC (BD), HLA DR,DQ,DP-PE(BD), β-2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC (BD).

Other antibody/label combinations that can be used include, but are notlimited to, CD45-PerCP (peridin chlorophyll protein); CD44-PE; CD19-PE;CD10-F (fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD);HLA-ABC-F; and the like.

The isolated placental cells provided herein can be assayed for CD117 orCD133 using, for example, phycoerythrin-Cy5 (PE Cy5) conjugatedstreptavidin and biotin conjugated monoclonal antibodies against CD117or CD133; however, using this system, the cells can appear to bepositive for CD117 or CD133, respectively, because of a relatively highbackground.

The isolated placental cells described herein can be labeled with anantibody to a single marker and detected and/sorted. Placental cells canalso be simultaneously labeled with multiple antibodies to differentmarkers.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Isolated placental cells can also be characterized and/or sorted basedon cell morphology and growth characteristics. For example, isolatedplacental cells can be characterized as having, and/or selected on thebasis of, e.g., a fibroblastoid appearance in culture. Isolatedplacental cells can also be characterized as having, and/or be selected,on the basis of their ability to form embryoid-like bodies. In oneembodiment, for example, isolated placental cells that are fibroblastoidin shape, express CD73 and CD105, and produce one or more embryoid-likebodies in culture are isolated from other placental cells. In anotherembodiment, OCT-4⁺ placental cells that produce one or moreembryoid-like bodies in culture are isolated from other placental cells.

In another embodiment, isolated placental cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as MESENCULT™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia).

Isolated placental cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Isolated placental cells can also be separated from other placentalcells using other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.5 Culture of Isolated Placental Cells

5.5.1 Culture Media

Isolated placental cells, or populations of isolated placental cells, orcells or placental tissue from which placental stem cells grow out, canbe used to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (e.g.,native or denatured), gelatin, fibronectin, ornithine, vitronectin, andextracellular membrane protein (e.g., MATRIGEL® (BD Discovery Labware,Bedford, Mass.)).

Isolated placental cells can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture ofcells, e.g., stem cells. Preferably, the culture medium comprises serum.The isolated placental cells can be cultured in, for example, DMEM-LG(Dulbecco's Modified Essential Medium, low glucose)/MCDB 201 (chickfibroblast basal medium) containing ITS (insulin-transferrin-selenium),LA+BSA (linoleic acid-bovine serum albumin), dextrose, L-ascorbic acid,PDGF, EGF, IGF-1, and penicillin/streptomycin; DMEM-HG (high glucose)comprising 1% to 20% fetal bovine serum (FBS); DMEM-HG comprising 15%FBS; IMDM (Iscove's modified Dulbecco's medium) comprising 10% FBS, 10%horse serum, and hydrocortisone; M199 comprising 10% FBS, EGF, andheparin; α-MEM (minimal essential medium) comprising 10% FBS, GLUTAMAX™and gentamicin; DMEM comprising 10% FBS, GLUTAMAX™ and gentamicin, etc.

Other media in that can be used to culture placental cells include DMEM(high or low glucose), Eagle's basal medium, Ham's F10 medium (F10),Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

The isolated placental cells can be cultured in standard tissue cultureconditions, e.g., in tissue culture dishes or multiwell plates. Theisolated placental cells can also be cultured using a hanging dropmethod. In this method, isolated placental cells are suspended at about1×10⁴ cells per mL in about 5 mL of medium, and one or more drops of themedium are placed on the inside of the lid of a tissue culturecontainer, e.g., a 100 mL Petri dish. The drops can be, e.g., singledrops, or multiple drops from, e.g., a multichannel pipetter. The lid iscarefully inverted and placed on top of the bottom of the dish, whichcontains a volume of liquid, e.g., sterile PBS sufficient to maintainthe moisture content in the dish atmosphere, and the stem cells arecultured.

In one embodiment, the isolated placental cells are cultured in thepresence of a compound that acts to maintain an undifferentiatedphenotype in the isolated placental cell. In a specific embodiment, thecompound is a substituted 3,4-dihydropyridimol[4,5-d]pyrimidine. In amore specific embodiment, the compound is a compound having thefollowing chemical structure:

The compound can be contacted with isolated placental cells, or apopulation of isolated placental cells, at a concentration of, forexample, between about 1 μM to about 10 μM.

5.5.2 Expansion and Proliferation of Placental Cells

Once an isolated placental cell, or population of isolated placentalcell (e.g., a placental cell or population of placental cells separatedfrom at least 50% of the placental cells with which the stem cell orpopulation of stem cells is normally associated in vivo), the cell orpopulation of cells can be proliferated and expanded in vitro. Forexample, a population of the isolated placental cells can be cultured intissue culture containers, e.g., dishes, flasks, multiwell plates, orthe like, for a sufficient time for the cells to proliferate to 70-90%confluence, that is, until the cells and their progeny occupy 70-90% ofthe culturing surface area of the tissue culture container.

The isolated placental cells can be seeded in culture vessels at adensity that allows cell growth. For example, the cells may be seeded atlow density (e.g., about 1,000 to about 5,000 cells/cm²) to high density(e.g., about 50,000 or more cells/cm²). In a preferred embodiment, thecells are cultured in the presence of about 0 to about 5 percent byvolume CO₂ in air. In some preferred embodiments, the cells are culturedat about 2 to about 25 percent O₂ in air, preferably about 5 to about 20percent O₂ in air. The cells preferably are cultured at about 25° C. toabout 40° C., preferably 37° C. The cells are preferably cultured in anincubator. The culture medium can be static or agitated, for example,using a bioreactor. Placental cells, e.g., placental stem cells orplacental multipotent cells, preferably are grown under low oxidativestress (e.g., with addition of glutathione, ascorbic acid, catalase,tocopherol, N-acetylcysteine, or the like).

Once a confluence of less than about 100%, for example 70%-90%, isobtained, the cells may be passaged. For example, the cells can beenzymatically treated, e.g., trypsinized, using techniques well-known inthe art, to separate them from the tissue culture surface. Afterremoving the cells by pipetting and counting the cells, about10,000-100,000 cells/cm², preferably about 50,000 cells/cm², arepassaged to a new culture container containing fresh culture medium.Typically, the new medium is the same type of medium from which theisolated placental cells were removed. The isolated placental cells canbe passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or20 times, or more.

Production of a Placental Cell Bank

Isolated cells from postpartum placentas, e.g., the isolated placentalcells described in Section 5.3, above, can be cultured in a number ofdifferent ways to produce a set of lots, e.g., a set ofindividually-administrable doses, of isolated placental cells. Such lotscan, for example, be obtained from cells from placental perfusate orfrom cells from enzyme-digested placental tissue. Sets of lots ofplacental cells, obtained from a plurality of placentas, can be arrangedin a bank of isolated placental cells for, e.g., long-term storage.Generally, tissue culture plastic-adherent placental cells are obtainedfrom an initial culture of placental material to form a seed culture,which is expanded under controlled conditions to form populations ofcells from approximately equivalent numbers of doublings. Lots arepreferably derived from the tissue of a single placenta, but can bederived from the tissue of a plurality of placentas.

In one embodiment, placental cell lots are obtained as follows.Placental tissue is first disrupted, e.g., by mincing, digested with asuitable enzyme, e.g., collagenase (see Section 5.4.3, above). Theplacental tissue preferably comprises, e.g., the entire amnion, entirechorion, or both, from a single placenta, but can comprise only a partof either the amnion or chorion. The digested tissue is cultured, e.g.,for about 1-3 weeks, preferably about 2 weeks. After removal ofnon-adherent cells, high-density colonies that form are collected, e.g.,by trypsinization. These cells are collected and resuspended in aconvenient volume of culture medium, and are then used to seed expansioncultures. Expansion cultures can be any arrangement of separate cellculture apparatuses, e.g., a Cell Factory by NUNC™. Cells can besubdivided to any degree so as to seed expansion cultures with, e.g.,1×10³, 2×10³, 3×10³, 4×10³ 5×10³, 6×10³, 7×10³, 8×10³ 9×10³ 1×10⁴ 1×10⁴,2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, or 10×10⁴ stemcells. Preferably, from about 1×10³ to about 1×10⁴ cells/cm² are used toseed each expansion culture. The number of expansion cultures may begreater or fewer in number depending upon the particular placenta(s)from which the cells are obtained.

Expansion cultures are grown until the density of cells in culturereaches a certain value, e.g., about 1×10⁵ cells/cm². Cells can eitherbe collected and cryopreserved at this point, or passaged into newexpansion cultures as described above. Cells can be passaged, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 timesprior to use. A record of the cumulative number of population doublingsis preferably maintained during expansion culture(s). The cells can beexpanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38 or 40 doublings, or up to 60 doublings.Preferably, however, the number of population doublings, prior todividing the population of cells into individual doses, is between about15 and about 30. The cells can be culture continuously throughout theexpansion process, or can be frozen at one or more points duringexpansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 50 million cells per ml, and can comprise between about 10⁶ andabout 10¹⁰ cells in total.

In one embodiment, therefore, a placental cell bank can be made by amethod comprising: expanding primary culture placental cells from ahuman post-partum placenta for a first plurality of populationdoublings; cryopreserving said placental cells to form a Master CellBank; expanding a plurality of placental cells from the Master Cell Bankfor a second plurality of population doublings; cryopreserving saidplacental cells to form a Working Cell Bank; expanding a plurality ofplacental cells from the Working Cell Bank for a third plurality ofpopulation doublings; and cryopreserving said placental cells inindividual doses, wherein said individual doses collectively compose aplacental cell bank. In another specific embodiment, said primaryculture placental cells comprise placental cells from placentalperfusate. In another specific embodiment, said primary cultureplacental cells comprise placental cells from digested placental tissue.In another specific embodiment, said primary culture placental cellscomprise placental cells from placental perfusate and from digestedplacental tissue. In another specific embodiment, all of said placentalcells in said placental cell primary culture are from the same placenta.In another specific embodiment, the method further comprises the step ofselecting CD200⁺ or HLA-G⁺ placental cells or CD10⁺, CD34⁻, CD105⁺,CD200⁺ placental cells from said plurality of said placental cells fromsaid Working Cell Bank to form individual doses. In another specificembodiment, said individual doses comprise from about 10⁴ to about 10⁵placental cells. In another specific embodiment, said individual dosescomprise from about 10⁵ to about 10⁶ placental cells. In anotherspecific embodiment, said individual doses comprise from about 10⁶ toabout 10⁷ placental cells. In another specific embodiment, saidindividual doses comprise from about 10⁷ to about 10⁸ placental cells.In another specific embodiment, said individual doses comprise fromabout 10⁸ to about 10⁹ placental cells. In another specific embodiment,said individual doses comprise from about 10⁹ to about 10¹⁰ placentalcells.

The methods of making compositions comprising placental cells, e.g.,placental stem cells or placental multipotent cells, as provided herein,can be integrated into the construction of a placental cell bank at anystep as described above. In one embodiment, the pharmaceuticalcomposition is produced after the Master Cell Bank is produced, andduring production of one or more Working Cell Banks from said MasterCell Bank, or during expansion of placental cells from said Working CellBanks. For example, placental cells can be thawed from a Working CellBank and cultured for a plurality of population doublings. In oneembodiment, when a desired number of cells is generated, or a desirednumber of population doublings has taken place, the placental cells canbe collected, e.g., by centrifugation, and resuspended in a solutioncomprising, e.g., dextran 40, e.g., 5.5% dextran 40. In certainembodiments, the placental stem cells are collected a second time andresuspended in a solution comprising dextran and a cryopreservant, e.g.,a 5.5% dextran 40 solution comprising 10% HSA and 5% DMSO, andcryopreserved. The cryopreserved placental cells are thawed, e.g.,immediately before use, e.g., immediately before final production of thecomposition as described in Section 5.2, above.

The above methods of producing a composition comprising placental cells,e.g., placental stem cells or placental multipotent cells, can be usedonce in the production and/or use of a placental cell bank, e.g., ateach point at which the placental cells would be cryopreserved, or,e.g., at the point at which placental cells are prepared for individualadministration prior to final cryopreservation, and upon thawing priorto administration to an individual.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. If the mothertests positive for a tested pathogen, the entire lot from the placentais discarded. Such testing can be performed at any time duringproduction of placental cell lots, including before or afterestablishment of the initial cell culture, or during expansion culture.Pathogens for which the presence is tested can include, withoutlimitation, hepatitis A, hepatitis B, hepatitis C, hepatitis D,hepatitis E, human immunodeficiency virus (types I and II),cytomegalovirus, herpesvirus, and the like.

5.6 Preservation of Placental Cells

Isolated placental cells, e.g., the isolated placental multipotent cellsdescribed in Section 5.2, above, can be preserved, e.g., duringcollection, that is, placed under conditions that allow for long-termstorage, or conditions that inhibit cell death by, e.g., apoptosis ornecrosis, e.g., during collection or prior to production of thecompositions described herein, e.g., using the methods described herein.

Placental cells can be preserved using, e.g., a composition comprisingan apoptosis inhibitor, necrosis inhibitor and/or an oxygen-carryingperfluorocarbon, as described in related U.S. Application PublicationNo. 2007/0190042, the disclosure of which is incorporated herein byreference in its entirety. In one embodiment, a method of preserving apopulation of cells, to be used in the compositions comprising cellspresented herein, comprises contacting said population of cells with acell collection composition comprising an inhibitor of apoptosis and anoxygen-carrying perfluorocarbon, wherein said inhibitor of apoptosis ispresent in an amount and for a time sufficient to reduce or preventapoptosis in the population of cells, as compared to a population ofcells not contacted with the inhibitor of apoptosis. In a specificembodiment, said inhibitor of apoptosis is a caspase inhibitor. Inanother specific embodiment, said inhibitor of apoptosis is a JNKinhibitor. In a more specific embodiment, said JNK inhibitor does notmodulate differentiation or proliferation of said cells. In anotherembodiment, said cell collection composition comprises said inhibitor ofapoptosis and said oxygen-carrying perfluorocarbon in separate phases.In another embodiment, said cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon in anemulsion. In another embodiment, the cell collection compositionadditionally comprises an emulsifier, e.g., lecithin. In anotherembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 0° C. and about 25° C. at the time of contacting thecells. In another more specific embodiment, said apoptosis inhibitor andsaid perfluorocarbon are between about 2° C. and 10° C., or betweenabout 2° C. and about 5° C., at the time of contacting the cells. Inanother more specific embodiment, said contacting is performed duringtransport of said population of cells. In another more specificembodiment, said contacting is performed during freezing and thawing ofsaid population of cells.

Populations of placental cells can be preserved, e.g., by a methodcomprising contacting said population of cells with an inhibitor ofapoptosis and an organ-preserving compound, wherein said inhibitor ofapoptosis is present in an amount and for a time sufficient to reduce orprevent apoptosis in the population of cells, as compared to apopulation of cells not contacted with the inhibitor of apoptosis. In aspecific embodiment, the organ-preserving compound is UW solution(described in U.S. Pat. No. 4,798,824; also known as ViaSpan; see alsoSouthard et al., Transplantation 49(2):251-257 (1990)) or a solutiondescribed in Stern et al., U.S. Pat. No. 5,552,267. In anotherembodiment, said organ-preserving compound is hydroxyethyl starch,lactobionic acid, raffinose, or a combination thereof. In anotherembodiment, the cell collection composition additionally comprises anoxygen-carrying perfluorocarbon, either in two phases or as an emulsion.

In another embodiment of the method, placental stem cells are contactedwith a cell collection composition comprising an apoptosis inhibitor andoxygen-carrying perfluorocarbon, organ-preserving compound, orcombination thereof, during perfusion. In another embodiment, said cellsare contacted during a process of tissue disruption, e.g., enzymaticdigestion. In another embodiment, placental cells are contacted withsaid cell collection compound after collection by perfusion, or aftercollection by tissue disruption, e.g., enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, acell, or population of cells, is exposed to a hypoxic condition duringcollection, enrichment or isolation for less than six hours during saidpreservation, wherein a hypoxic condition is a concentration of oxygenthat is less than normal blood oxygen concentration. In a more specificembodiment, said population of cells is exposed to said hypoxiccondition for less than two hours during said preservation. In anothermore specific embodiment, said population of cells is exposed to saidhypoxic condition for less than one hour, or less than thirty minutes,or is not exposed to a hypoxic condition, during collection, enrichmentor isolation. In another specific embodiment, said population of cellsis not exposed to shear stress during collection, enrichment orisolation.

Placental cells can be cryopreserved, in general or in the specificmethods disclosed herein. In one embodiment, the cryopreservative usedto cryopreserve placental cells is DMSO. In another embodiment, thecryopreservative is propylene glycol, e.g., about 1.5 M propyleneglycol. The cryopreservative may also be, e.g., glycerol, ethyleneglycol, polyphenol (e.g., at about 30 to about 120 ppm) or the like Inother embodiments, the cryopreservative is fetal bovine serum, humanserum, or human serum albumin in combination with one or more of DMSO,trehalose, and dextran. In a specific embodiment, the cryopreservativeis human serum, DMSO, and trehalose, or is fetal bovine serum and DMSO.In certain embodiments, the placental cells are cryopreserved incryopreservation medium in small containers, e.g., ampoules. Placentalcells are preferably cooled at about 1° C./min during cryopreservation.A preferred cryopreservation temperature is about −80° C. to about −180°C., preferably about −125° C. to about −140° C. Cryopreserved cells canbe transferred to liquid nitrogen prior to thawing for use. In someembodiments, for example, once the ampoules have reached about −90° C.,they are transferred to a liquid nitrogen storage area. Cryopreservationcan also be done using a controlled-rate freezer. Cryopreserved cellspreferably are thawed at a temperature of about 25° C. to about 40° C.,preferably to a temperature of about 37° C.

5.7 Cell Containing Compositions

5.7.1 Compositions Comprising Placental Cells

The placental cells described herein, e.g., in Section 5.3, can compriseone or more of the placental cells, e.g., placental stem cells orplacental multipotent cells, described herein, wherein the cells havebeen isolated from a placenta, e.g., a human placenta. In anotherspecific embodiment, any of the foregoing compositions comprises amatrix. In a more specific embodiment, said matrix is athree-dimensional scaffold. In another more specific embodiment, saidmatrix comprises collagen, gelatin, laminin, fibronectin, pectin,ornithine, or vitronectin. In another more specific embodiment, thematrix is an amniotic membrane or an amniotic membrane-derivedbiomaterial. In another more specific embodiment, said matrix comprisesan extracellular membrane protein. In another more specific embodiment,said matrix comprises a synthetic compound. In another more specificembodiment, said matrix comprises a bioactive compound. In another morespecific embodiment, said bioactive compound is a growth factor,cytokine, antibody, or organic molecule of less than 5,000 daltons.

In another embodiment, a composition useful in the compositions, e.g.,pharmaceutical compositions, provided herein comprises mediumconditioned by any of the foregoing placental cells, or any of theforegoing placental cell populations. In a specific embodiment, any suchcomposition comprises a stem cell that is not derived from a placenta.In a more specific embodiment, said stem cell is an embryonic stem cell.In another more specific embodiment, said stem cell is a mesenchymalstem cell. In another more specific embodiment, said stern cell is abone marrow-derived stem cell. In another more specific embodiment, saidstem cell is a hematopoietic progenitor cell. In another more specificembodiment, said stem cell is a somatic stem cell. In an even morespecific embodiment, said somatic stem cell is a neural stem cell, ahepatic stem cell, a pancreatic stem cell, an endothelial stem cell, acardiac stem cell, or a muscle stem cell.

5.7.1.1 Pharmaceutical Compositions

Populations of isolated placental cells, or populations of cellscomprising the isolated placental cells, are contained within, or arecomponents of, a pharmaceutical composition. Isolated placental cellscan be prepared in a form that is easily administrable to an individual,e.g., placental perfusate cells or isolated placental cells that arecontained within a container suitable for medical use. Such a containercan be, for example, a syringe, sterile plastic bag, flask, jar, orother container from which the placental stem cell population can beeasily dispensed. For example, the container can be a blood bag or otherplastic, medically-acceptable bag suitable for the intravenousadministration of a liquid to a recipient. The container in certainembodiments one that allows for cryopreservation of the isolatedplacental cell population.

In one embodiment, the container is a container that facilitates, orallows, performance of one or more of the method steps described herein.For example, where the method of producing a composition comprisingcells comprises, e.g., the steps of (a) contacting said cells with asolution comprising dextran and human serum albumin (HSA) to produce acell-containing solution; (b) filtering the solution; (c) diluting saidcells to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to15×10⁶, or 1 to 10×10⁶ cells per milliliter with a first dilutionsolution comprising dextran; and (d) diluting said cells with a seconddilution solution comprising dextran but not comprising HSA, the cells,e.g., isolated placental cells or placental perfusate cells, can beplaced into a container, e.g., between steps (c) and (d), wherein thecontainer is a container that, e.g., facilitates cryopreservation and/orfacilitates delivery of the cells to an individual in need of the cells,or the like. In certain embodiments, said diluting is to no more thanabout 15×10⁶ cells per milliliter. In certain embodiments, said dilutingis to no more than about 10±3×10⁶ cells per milliliter. In other certainembodiments, if the number of cells is less than about 10±3×10⁶ cellsper milliliter, filtration is optional.

For example, the isolated placental cells can be cryopreserved in, e.g.,a bag, e.g., a blood bag or similar bag, and thawed and finally dilutedin the same bag. In another embodiment, wherein the method of producinga composition comprising cells comprises, e.g., (a) centrifuging aplurality of cells, e.g., placental perfusate cells or isolatedplacental cells to collect the cells; (b) resuspending the cells in 5.5%dextran 40; (c) centrifuging the cells to collect the cells; (d)resuspending the cells in a 5.5% dextran 40 solution that comprises 10%HSA; (e) filtering the cells through a 70 μM to 100 μM filter; (f)diluting the cells in 5.5% dextran 40, 10% HSA, and 5% DMSO to no morethan about 10±3×10⁶ cells/mL; (g) cryopreserving the cells; (h) thawingthe cells; and (i) diluting the cells 1:1 to 1:11 with 10% dextran 40 toproduce said pharmaceutical composition, placement of the cells in acontainer that, e.g., facilitates cryopreservation and/or administrationof the cells to an individual in need thereof can be performed, e.g., atany step after step (e). In certain embodiments, said diluting in step(I) is to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1 to15×10⁶, or 1 to 10×10⁶ cells per milliliter. In certain embodiments,said diluting in step (f) is to no more than about 15×10⁶ cells permilliliter. In other certain embodiments, if the number of cells is lessthan about 10±3×10⁶ cells per milliliter, filtration is optional. In aspecific embodiment, for example, cells, e.g., isolated placental cellsor placental perfusate cells, can be placed into the container afterfiltration, then, in the container, diluted, cryopreserved, thawed,and/or finally diluted prior to administration to the individual.

Isolated placental cells in the compositions, e.g., pharmaceuticalcompositions, provided herein, can comprise placental cells derived froma single donor, or from multiple donors. The isolated placental cellscan be completely HLA-matched to an intended recipient, or partially orcompletely HLA-mismatched.

Thus, in one embodiment, isolated placental cells in the compositionsprovided herein are administered to an individual in need thereof. In aspecific, said isolated placental cells are administeredintramuscularly, intradermally, intraperitoneally, intra-arterially,subcutaneously, intravenously or intraocularly. In one embodiment,isolated placental cells in the compositions provided herein areadministered to an individual in need thereof in the form of acomposition comprising isolated placental cells in a container. Inanother specific embodiment, the container is a bag, flask, or jar. Inmore specific embodiment, said bag is a sterile plastic bag. In a morespecific embodiment, said bag is suitable for, allows or facilitatesintravenous administration of said isolated placental cells, e.g., byintravenous infusion, bolus injection, or the like. The bag can comprisemultiple lumens or compartments that are interconnected to allow mixingof the isolated placental cells and one or more other solutions, e.g., adrug, prior to, or during, administration. In another specificembodiment, prior to cryopreservation, the solution comprising theisolated placental cells comprises one or more compounds that facilitatecryopreservation of the combined cells. In another specific embodiment,said isolated placental cells are contained within aphysiologically-acceptable aqueous solution. In a more specificembodiment, said physiologically-acceptable aqueous solution is a 0.9%NaCl solution. In another specific embodiment, said isolated placentalcells comprise placental cells that are HLA-matched to a recipient ofsaid cell population. In another specific embodiment, said combinedcells comprise placental cells that are at least partiallyHLA-mismatched to a recipient of said cell population. In anotherspecific embodiment, said placental cells are derived from a pluralityof donors.

The isolated placental cells in the pharmaceutical composition can beany of the isolated placental cells described herein. In a specificembodiment, the isolated placental cells described herein are CD10⁺,CD34⁻, CD105⁺, CD200⁺, cells that are contained within a container. In aspecific embodiment, the isolated placental cells described herein areCD200⁺, HLA-G⁺ cells that are contained within a container. In anotherspecific embodiment, the isolated placental cells are CD73⁺, CD105⁺,CD200⁺ cells that are contained within a container. In another specificembodiment, the isolated placental cells are CD200⁺, OCT-4⁺ cells thatare contained within a container. In another specific embodiment, theisolated placental cells are CD73⁺, CD105⁺ cells that are containedwithin a container, wherein said cells facilitate the formation of oneor more embryoid-like bodies when cultured with a population ofplacental cells under conditions that allow for the formation ofembryoid-like bodies. In another specific embodiment, the isolatedplacental cells are CD73⁺, CD105⁺, HLA-G⁺ cells that have beencryopreserved, and are contained within a container. In another specificembodiment, the isolated placental cells are OCT-4⁺ cells that arecontained within a container, wherein said cells facilitate theformation of one or more embryoid-like bodies when cultured with apopulation of placental cells under conditions that allow for theformation of embryoid-like bodies. In a specific embodiment of any ofthe foregoing placental cells, said container is a bag.

In various specific embodiments, said container comprises about; atleast, or at most 1×10⁶ isolated placental cells or placental perfusatecells, 5×10⁶ isolated placental cells or placental perfusate cells,1×10⁷ isolated placental cells or placental perfusate cells, 5×10⁷isolated placental cells or placental perfusate cells, 1×10⁸ isolatedplacental cells or placental perfusate cells, 5×10⁸ isolated placentalcells or placental perfusate cells, 1×10⁹ isolated placental cells orplacental perfusate cells, 5×10⁹ isolated placental cells or placentalperfusate cells, or 1×10¹⁰ isolated placental cells or placentalperfusate cells. In other embodiments, a single unit dose of isolatedplacental cells or placental perfusate cells can comprise, in variousembodiments, about, at least, or no more than 1×10⁵, 5×10⁵, 1×10⁶,5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹or more isolated placental cells or placental perfusate cells. In otherembodiments, a container or dose of isolated placental cells orplacental perfusate cells comprises 1×10⁵ to 5×10⁵ isolated placentalcells or placental perfusate cells, 5×10⁵ to 1×10⁶ isolated placentalcells or placental perfusate cells, 1×10⁶ to 5×10⁶ isolated placentalcells or placental perfusate cells, 5×10⁶ to 1×10⁷ isolated placentalcells or placental perfusate cells, 1×10⁷ to 5×10⁷ isolated placentalcells or placental perfusate cells, 5×10⁷ to 1×10⁸ isolated placentalcells or placental perfusate cells, 1×10⁸ to 5×10⁸ isolated placental orplacental perfusate cells, 5×10⁸ to 1×10⁹ isolated placental orplacental perfusate cells, 1×10⁹ to 5× isolated placental cells orplacental perfusate cells, 5×10⁹ to 1×10¹⁰ isolated placental cells orplacental perfusate cells, 1×10¹⁰ to 5×10¹⁰ isolated placental cells orplacental perfusate cells, 5×10¹⁰ to 1×10¹¹ isolated placental cells orplacental perfusate cells, or more isolated placental or placentalperfusate cells. In a preferred embodiment, the pharmaceuticalcomposition comprises a sufficient number of isolated placental cells orplacental perfusate cells to administer about 2×10⁷ to about 10×10⁷cells per kilogram of a recipient.

In other specific embodiments of any of the foregoing cryopreservedpopulations, said cells have been passaged about, at least, or no morethan 5 times, no more than 10 times, no more than 15 times, or no morethan 20 times. In another specific embodiment of any of the foregoingcryopreserved cells, said cells have been expanded within saidcontainer.

Pharmaceutical compositions comprising the placental stem cellsdescribed herein can comprise any, or any combination, of the isolatedplacental cell populations, or isolated placental cell types, describedelsewhere herein. The pharmaceutical compositions can comprise fetal,maternal, or both fetal and maternal placental cells, e.g., placentalstem cells or placental multipotent cells. The pharmaceuticalcompositions provided herein can further comprise isolated placentalcells obtained from a single individual or placenta, or from a pluralityof individuals or placentae.

The pharmaceutical compositions provided herein comprise populations ofcells that comprise 50% viable cells or more (that is, at least 50% ofthe cells in the population are functional or living). Preferably, atleast 60% of the cells in the population are viable. More preferably, atleast 70%, 80%, 90%, 95%, or 99% of the cells in the population in thepharmaceutical composition are viable.

In one embodiment, the pharmaceutical composition comprises isolatedplacental cells that are substantially, or completely, non-maternal inorigin, that is, have the fetal genotype; e.g., at least about 90%, 95%,98%, 99% or about 100% are non-maternal in origin. For example, in oneembodiment a pharmaceutical composition comprises a population ofisolated placental cells that are CD200⁺ and HLA-G⁺; CD73⁺, CD105⁺, andCD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺ and CD105⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said population of isolatedplacental cell when said population of placental cells is cultured underconditions that allow the formation of an embryoid-like body; or OCT-4⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said population of isolatedplacental cell when said population of placental cells is cultured underconditions that allow the formation of an embryoid-like body; or acombination of the foregoing, wherein at least 70%, 80%, 90%, 95% or 99%of said isolated placental cells are non-maternal in origin. In anotherembodiment, a pharmaceutical composition comprises a population ofisolated placental cells that are CD10⁺, CD105⁺ and CD34⁻; CD10⁺,CD105⁺, CD200⁺ and CD34⁻; CD10⁺, CD105⁺, CD200⁺, CD34 and at least oneof CD90⁺ or CD45⁻; CD10⁺, CD90⁺, CD105⁺, CD200⁺, CD34⁻ and CD45⁻; CD10⁺,CD90⁺, CD105⁺, CD200⁺, CD34⁻ and CD45⁻; CD200⁺ and HLA-G⁺; CD73⁺,CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁺; CD73⁺and CD105⁺ and facilitate the formation of one or more embryoid-likebodies in a population of placental cells comprising said isolatedplacental cells when said population of placental cells is culturedunder conditions that allow the formation of an embryoid-like body;OCT-4⁺ and facilitate the formation of one or more embryoid-like bodiesin a population of placental cells comprising said isolated placentalcells when said population of placental cells is cultured underconditions that allow the formation of an embryoid-like body; or one ormore of CD117⁻, CD133⁻, KDR⁻, CD80⁻, CD86⁻, HLA-A,B,C⁺, HLA-DP,DQ,DR⁻and/or PDL1⁺; or a combination of the foregoing, wherein at least 70%,80%, 90%, 95% or 99% of said isolated placental cells are non-maternalin origin. In a specific embodiment, the pharmaceutical compositionadditionally comprises a stem cell that is not obtained from a placenta.

The pharmaceutical compositions provided herein can comprise one or morecompounds that, e.g., facilitate engraftment (e.g., anti-T-cell receptorantibodies, an immunosuppressant, or the like); stabilizers such asalbumin, dextran 40, gelatin, hydroxyethyl starch, Plasmalyte, and thelike.

5.7.2 Compositions Comprising Hematopoietic Placental Cells or PlacentalPerfusate Cells

In certain embodiments of the compositions provided herein, the isolatedplacental cells are CD34⁺ placental stem cells, e.g., hematopoieticplacental cells or progenitor cells. Such cells are obtainable fromplacental tissue, e.g., from a placenta that has been drained of cordblood and perfused to remove residual blood. In certain embodiments, theCD34⁺ placental stem cells are CD38⁺. In certain embodiments, the CD34⁺isolated placental cells are CD38⁻. In certain other embodiments, theCD34⁺ isolated placental cells are CD45⁺. In a specific embodiment, theisolated placental cells are CD34⁺, CD38⁻ and CD45⁺. In certainembodiments, the cells are hematopoietic cells. In a more specificembodiment, the placental CD34⁺ cells are hematopoietic cells. Incertain embodiments, the CD34⁺ cells or hematopoietic cells are obtainedfrom placental perfusate. In certain embodiments, the CD34⁺ cells orhematopoietic cells are obtained enzymatic digestion or physicaldisruption of placental tissue. The CD34⁺ cells and hematopoietic cellscan be obtained from a single placenta, or from more than one placenta.

In certain embodiments, the cells of the compositions provided herein,formulated by the methods provided herein, are cells obtained fromplacental perfusate. As used herein, “cells obtained from placentalperfusate” includes total nucleated cells obtained from, e.g., isolatedfrom, placental perfusate, a subset of nucleated cells obtained fromplacental perfusate, or cells cultured or proliferated from cellsobtained directly from placental perfusate. Placental perfusate may beobtained from a placenta that has been drained of cord blood andperfused to remove residual blood, prior to perfusion to obtainplacental cells. Placental perfusate may be obtained from a placentathat has been drained of cord blood but not perfused to remove residualblood. Placental perfusate may be obtained from a placenta that hasneither been drained of cord blood nor perfused to remove residualblood. In the latter two embodiments, the placental cells, e.g.,nucleated cells from placental perfusate, for example, total nucleatedcells from placental perfusate, comprise nucleated cells from placentalblood and/or cord blood. the placental perfusate cells can be obtainedfrom a single placenta, or from more than one placenta.

5.7.3 Immortalized Placental Cell Lines

Mammalian placental cells can be conditionally immortalized bytransfection with any suitable vector containing a growth-promotinggene, that is, a gene encoding a protein that, under appropriateconditions, promotes growth of the transfected cell, such that theproduction and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1aadenovirus or E7 protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells, in one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV*−1), a minimal promoter from human cytomegalovirus fused totet operator sequences, tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed within the present methods. Cells carrying neo^(R) may beselected by means known to those of ordinary skill in the art, such asthe addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is a polyomithine/lamininsubstrate, consisting of tissue culture plastic coated withpolyornithine (10 μg/mL) and/or laminin (10 ng/mL), a polylysine/lamininsubstrate or a surface treated with fibronectin. Cultures are then fedevery 3-4 days with growth medium, which may or may not be supplementedwith one or more proliferation-enhancing factors.Proliferation-enhancing factors may be added to the growth medium whencultures are less than 50% confluent.

The conditionally-immortalized placental cell lines can be passagedusing standard techniques, such as by trypsinization, when 80-95%confluent. Up to approximately the twentieth passage, it is, in someembodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman placental cell line prepared as described above. In general, suchclonal cell lines may be isolated using standard techniques, such as bylimit dilution or using cloning rings, and expanded. Clonal cell linesmay generally be fed and passaged as described above.

Conditionally-immortalized human placental cell lines, which may, butneed not, be clonal, may generally be induced to differentiate bysuppressing the production and/or activity of the growth-promotingprotein under culture conditions that facilitate differentiation. Forexample, if the gene encoding the growth-promoting protein is under thecontrol of an externally-regulatable promoter, the conditions, e.g.,temperature or composition of medium, may be modified to suppresstranscription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

5.7.4 Kits

In another aspect, further provided herein are kits for the productionand/or administration of the isolated placental cell-containingcompositions of the present invention.

In one embodiment, provided herein is a kit comprising, in separatecontainers, one or more of a solution comprising dextran, e.g., dextran40, a solution comprising human serum albumin (HSA), and acryopreservant. In a specific embodiment, the kit comprises a pluralityof isolated placental cells, e.g., cryopreserved isolated placentalcells. In a specific embodiment, the kit comprises a containercomprising a solution comprising 5.5% dextran 40 (w/v) and 10% HSA(w/v). In another specific embodiment, the kit comprises a containercomprising a solution comprising 5.5% dextran 40, 10% HSA and 5% DMSO.In another specific embodiment, the kit comprises a container comprisinga solution of 10% dextran 40.

In another embodiment, the kit comprises a filter, or plurality offilters, suitable for filtering cell suspensions. In specificembodiments, one or more of the filters in the kit comprise poresbetween about 50 μM in diameter to about 150 μM in diameter. In morespecific embodiment, the filter is a 70 μM filter. In another specificembodiment, the filter is a 100 μM filter.

In another embodiment, the kit comprises one or more articles ofglassware or plasticware suitable for the production or use of one ofthe compositions described herein. For example, the kit can comprise,e.g., a plastic bag suitable for the cryopreservation or dilution ordelivery of a cell suspension, e.g., a suspension of isolated placentalcells. In another embodiment, the kit comprises (1) a plurality ofisolated placental cells, e.g., placental stem cells or placentalmultipotent cells, e.g., in one or more vials; (2) plasticwaresufficient to culture said isolated placental cells for, e.g., 2-3passages; (3) a container comprising a solution comprising 5.5% dextran40 (w/v) and 10% HSA (w/v); (4) a container comprising a solutioncomprising 5.5% dextran 40, 10% HSA and 5% DMSO; (5) a containercomprising a 10% dextran 40 solution; and (6) one or more filterscomprising pores between about 50 μM in diameter to about 150 μM indiameter, wherein the filters are suitable for filtering solutionscomprising cells.

6. EXAMPLES 6.1 Example 1: Improved Method of Producing AdministrableCompositions Comprising Isolated Placental Cells

This Example demonstrates formulation of human CD34⁻, CD10⁺, CD105⁺,CD200⁺ isolated placental cells both before and after cryopreservation,to produce homogenous, high viability isolated placental cells foradministration to humans or animals. The resulting composition comprisesisolated placental cells that exhibit high viability and no macro cellclumps over at least a 4 hour period post-thaw. An acute dosing mousestudy demonstrated that NOD/SCID mice tolerated a maximum dose of atleast 1.5 million cells per mouse (approximately 75 million cells per kgassuming an average weight of 20 grams) using the final formulationdescribed below by intravenous infusion administration without anycardiac or pulmonary toxicity (evidenced by labored breathing, circling,and ataxia), and up to 250 million cells per kg subcutaneously, animprovement over previous formulations. While the following exampledescribes formulation of isolated placental cells expressing particularsurface markers, the results presented herein indicate that the methodsand formulations can be used, and are compatible, with other cells,e.g., mammalian cells expressing different surface markers.

Cell Clump Assays

Cell clumps (aggregations) were classified as macro cell clumps or microcell clumps. Macro cell clumps and micro cell clumps were identified, ifpresent, according to the following procedures.

Macro Cell Clump Assay

Cells were thawed in a container in a 37° C. water bath until only atiny piece of ice remained in the container. Cells were drawn from thecontainer using a syringe fitted with a 16 gauge needle, and the cellswere dispensed from the container into a 50 mL conical tube. Thepresence or absence of macro cell clumps was assessed by visualinspection.

Micro Cell Clump Assay

Cells were counted to determine cell concentration. Cells were thendiluted to 4×10⁶ cell/ml with 10% dextran 40, and 50 μL of the cells, 40μL of 10% dextran 40, and 10 μL of a 40 μM measurement bead solution wasadded to a 1.7 mL microcentrifuge tube. The contents of the tube weremixed gently. Ten μL of the mixed sample was placed on a glass slide andcovered with a cover slip. All micro cell clumps comprising three ormore cells were counted.

Placental cells used were initially obtained from a cell bank thatcontained populations of adherent placental cells, as described herein,that have undergone 4-6 passages prior to cryopreservation.

Data from comparison of in vivo injection compatible buffers, using twodifferent isolated placental cell lines, indicated that phosphatebuffered saline (PBS), Plasmalyte A and Dulbecco's Modified Eagle'sMedium (DMEM) with the supplement of 1% human serum albumin (HSA) areall able to maintain high viability of cells after 5 hours post-thaw.The final cell concentration in the buffer was 25×10⁶ cells/ml.Plasmalyte A was selected as promoting the highest viability of cellsfor the buffers tested. As demonstrated in Table 10a and Table 10b, highviability was maintained for several hours post-thaw; furthermore,nominal phenotypes were not changed over 3 hours post-thaw. Viability ofthe cells in the post-thaw formulation was not significantly affectedafter passage of the cells through a 26-gauge needle. A determination ofcell clumping in these two experiments was not possible due to the smallsample volume used.

Based on the data in Table 1a and Table 1b, the post-thaw formulationwas finalized as follows: Cells were thawed at 37° C. in a water bath,and immediately diluted 1:1 (v/v) with thawing buffer (2.5% HSA+5%Dextran 40). The cells were then centrifuged at 400 g for 5 minutes, andresuspended in Plasmalyte A+1% HSA.

TABLE 1a Post-thaw viability and phenotype of placental cells inPlasmalyte A + 1% HSA without a syringe/needle test. 0 hour 1 hour 3hour 5 hour Viability 98.0% 97.1% 92.4% 93.8% CD105+/200+ 84.3% 87.9%

TABLE 1b Post-thaw viability and phenotype of placental cells inPlasmalyte A + 1% HSA with a syringe/needle test. 0 hour 1 hour 3 hour 5hour Viability 98.0% 97.2% 93.7% 97.3% CD105+/200+ 87.3% 87.3%

Mouse Biodistribution Study

The above post-thaw formulation was applied in a pilot mousebiodistribution study. Macro cell clumps were observed during post-thawcell preparation, especially after the addition of Plasmalyte. With thiscell preparation, acute pulmonary toxicity was observed at a dose of 1million cells per mouse (around 20 g) by two repeat intravenousinfusions of 0.5 million cells each. These two observations promptedfurther investigation of the placental cell formulation.

Based upon the observations from a pilot mouse biodistribution studythat significant cell clumps were induced after the addition ofPlasmalyte A, but not Dextran 40, Dextran 40 was used for this study asa dilution medium, along with Plasmalyte A, HSA and PBS.

TABLE 2 Cell clumps ranking at 4 hours post-thaw. Lower numbers indicatefewer clumps. 5% Dextran 5% Dextran 40 + 40 + 5% Dextran Medium 10% HSA2.5% HSA 40 PBS Plasmalyte A Cell 1 1 2 3 3 clump rank

Cells, previously frozen in Plasmalyte A+10% HSA+5% DMSO, were thawed ina 37° C. water bath, and diluted 1:7 with the respective buffers. Thedata in table 2 shows that Dextran 40 with HSA induced the fewest cellclumps among the media tested.

Cell aggregates were observed immediately post-thaw in variousformulations. Addition of a filtration step, wherein post-thaw cellswere filtered through a 100 μm filter, eliminated cell aggregates. Twolots of placental cells were tested for the effect of filtration incombination with specific diluents. No macro cell clumps were formedpost-filtration when cells were 1:1 diluted with 10% Dextran 40 over atime period of 4 hours post-thaw. See Tables 3a-3c. In addition,viability remained high. Similar results were observed across severaldifferent lots of placental cells.

TABLE 3a Macro cell clump LOT 1 LOT 2 Existing macro cell Yes, largesheets Yes, small dots clump in bag post- thaw Newly formed Plasmalyte A5% Dextran 40 Plasmalyte A 5% Dextran 40 macro cell clump Yes No Yes,but much No post-filtration fewer than LOT 1

TABLE 3b Micro cell clump post-filtration in Dextran 40 LOT 1 LOT 2 3-5cell clump 1210 416 (clumps/10⁶ cells) 5 cell clump 491 119 (clumps/10⁶cells)

TABLE 3c Viability post-filtration in Dextran 40 and Plasmalyte A LOT 1LOT 2 Dextran 40 Plamalyte A Dextran 40 Plasmalyte A 0 hr 91.5% 92.4%94.2% 94.5% 2 hr 92.5% 93.9% 4 hr 91.8% 94.7% 94.7% 93.3%

On the basis of the foregoing studies, the post-thaw formulationplacental cells was simplified to the following procedure: Cells werethawed at 37° C. water bath for up to 3 minutes, then filtered through a100 μM strainer. The strained cells were then diluted 1:1 (v:v) with 10%dextran 40.

Pre-Freeze Formulation

Cell clumps were also observed in the pre-freeze phase of cellprocessing. Freezing medium, freezing cell density and a suspensionfiltration step were studied to reduce and/or eliminate cell aggregatesprior to freezing.

Freezing Medium

Based upon the success of Dextran 40 in the post-thaw formulation,above, Dextran 40 was compared with Plasmalyte as a freezing medium.Cells from LOT 3 were frozen at a concentration of 17 million cells permL in either Plasmalyte A+10% HSA+5% DMSO or in 5% Dextran 40+10% HSA+5%DMSO. The presence of cell clumps was evaluated as follows:

TABLE 4a Comparison of Plasmalyte and Dextran 40 as freezing medium -Macro cell clumps Plasmalyte A Dextran 40 Macro cell clump More thanDextran 40 Fewer than Plasmalyte A Cell loss after 20% 3% filtrationwith 100 μm strainer

TABLE 4b Comparison of Plasmalyte A and Dextran 40 as freezing medium -Micro cell clumps Plasmalyte A Dextran 40 3-5 cell clump 1893 523(clumps/10⁶ cells) >5 cell clump 929 205 (clumps/10⁶ cells)

TABLE 4c Comparison of Plasmalyte A and Dextran 40 as freezing medium -Viability Plasmalyte Dextran 40 0 hour post-thaw 89.9% 90.8% 4 hourpost-thaw 89.2% 91.3%

The use of Dextran 40 in these experiments resulted in fewer macro cellclumps, fewer micro cell clumps, and higher cell viability than the useof Plasmalyte A. On the basis of these results, Dextran 40 was selectedas the freezing medium.

Freezing Cell Density and Pre-Freeze Filtration

In order to eliminate macro cell clumps and minimize micro cell clumps,cell density and pre-freeze filtration were examined as follows:

TABLE 5a The effect of freezing cell density and pre-freeze filtrationon cell clump formation. Condition 1 Condition 2 Condition 3 Condition 4Filtration Yes, 70 μm No Yes, 70 μm No strainer strainer Concentration20 × 10⁶ 20 × 10⁶ 5 × 10⁶ 5 × 10⁶ Freezing 5% Dextran 40 + 5% Dextran40 + 5% Dextran 40 + 5% Dextran 40 + medium 10% HSA + 5% 10% HSA + 5%10% HSA + 5% 10% HSA + 5% DMSO DMSO DMSO DMSO Concentration: cells permilliliter.

TABLE 5b The effect of freezing cell density and pre-freeze filtrationon macro cell clump formation. Condition 1 Condition 2 Condition 3Condition 4 Pre-freeze No Yes No Yes filtration Post-thaw No Yes No Yesfiltration No: No macro clump formation. Yes: Macro clump formation.

TABLE 5c The effect of freezing cell density and pre-freeze filtrationon micro cell clump (clumps per 10⁶ cells) Condition 1 Condition 2Condition 3 Condition 4 3-5 >5 cell 3-5 cell >5 cell 3-5 cell >5 cell3-5 cell >5 cell micro micro micro micro micro micro micro micro cellcell cell cell cell cell cell cell clumps clumps clumps clumps clumpsclumps clumps clumps Pre-freezing 188 0 291 194 231 46 237 172 Post-thaw688 375 631 655 231 116 323 366

The results shown in Tables 5b and 5c clearly show that pre-freezefiltration eliminated post-thaw macro cell clump formation. The dataalso indicate that samples with high cell concentration, e.g., 20×10⁶cells/ml, have more potential to form micro cell clumps post-thaw. As aresult, the effect of freezing cell density on cell clump formation wasexamined further.

TABLE 6a Effect of freezing cell density on cell clump formation LOT 4Condition 1 Condition 2 Condition 3 Condition 4 Concentration 15 × 10⁶10 × 10⁶ 7.5 × 10⁶ 5 × 10⁶ Filtration 70 μm strainer 70 μm strainer 70μm strainer 70 μm strainer Freezing 5% Dextran 40 + 5% Dextran 40 + 5%Dextran 40 + 5% Dextran 40 + medium 10% HSA + 5% 10% HSA + 5% 10% HSA +5% 10% HSA + 5% DMSO DMSO DMSO DMSO Concentration: number of cells permilliliter.

TABLE 6b Effect of freezing cell density on macro cell clump formationCondition 1 Condition 2 Condition 3 Condition 4 Pre- Yes (1 clump) No NoNo freezing Post-thaw Yes (3 clumps) Yes (1 clump) No No 0 h Post-thawYes (3 clumps) Yes (1 clump) No No 4 h No: No macro cell clumpformation. Yes: Macro cell clump formation

TABLE 6c The effect of freezing cell density on micro cell clump (clumpsper 10⁶ cells) Condition 1 Condition 2 Condition 3 Condition 4 3-5cell >5 cell 3-5 cell >5 cell 3-5 cell >5 cell 3-5 cell >5 cellPre-freezing 141 70 130 65 83 33 103 34 Post-thaw 0 h 271 193 111 44 10234 108 46 Post-thaw 4 h 298 212 105 53 122 44 97 55

The results in Tables 6b and 6c demonstrate that freezing density at7.5×10⁶/ml or less does not induce post-thaw macro cell clumps.Furthermore, at concentrations up to 10×10⁶ cells/ml, the number ofmicro cell clumps is reasonably low, at less than 200 micro cellclumps/million cells.

Based on the above results, the pre-freeze and post-thaw formulationswere generated as follows. For pre-freeze formulation, placental cellsfrom liquid culture were centrifuged at 220×g for 5 minutes, andresuspended in 5% Dextran 40 to about 7.5×10⁶ cells/mL. The cells werecentrifuged at 400×g for 10 minutes, then resuspended in 6% Dextran 40and 10% HSA to about 7.5×10⁶ cells/mL. The resuspended cells were thenpassed through a 70 μM filter by gravity. Filtered cells were thendiluted in 5% Dextran 40, 10% HSA, and 5% DMSO to a concentration ofabout 7.5×10⁶ cells per mL. The diluted cells were placed in cryo-bags,frozen, and stored under vapor phase nitrogen. For post-thawformulation, frozen cells were thawed in a 37° C. water bath, thendiluted with 10% dextran 40 at various volume ratios from 1:1 to 1:5 ofcell-containing buffer:dextran 40

Assessment of Placental Cell Formulation

1. No Post-Thaw Macro Cell Clump Formation

Five lots of placental cells were produced using the above formulationmethod (including LOT 11 and LOT 12, described below). Macro cell clumpspost-thaw were not observed in any of the five lots, and the number ofmicro cell clumps remained low.

2. No Impact on Phenotype

The phenotype of the placental cells was not affected by the improvedformulation. More than 90% of cells in the formulation remained CD10⁺,CD34⁻, CD105⁺ and CD200⁺.

3. GLP Mouse Studies

LOT 12 cells were used for a mouse biodistribution study. Placentalcells were administered in the above formulation as a single and/orrepeat intravenous tail vein injection to both male and female NOD-SCIDor male C57BL/10SgSnAi-Rag2(tmi)γc(tmi) mice. The mice were sacrificedat 4, 14, 28 or 47 days post-treatment and samples of lung, liver,heart, kidneys, spleen, adrenal glands, bone marrow, and brain wereprocessed and analyzed by Q-PCR for the presence of the hTERT DNAsequence; hTERT is the human telomerase reverse transcriptase gene.Following i.v. administration, human DNA was detected in isolated totalDNA from samples of lung, brain, heart and/or liver in mice that weresacrificed at 4 days after dosage administration. The highest levels ofDNA were detected in the lung. Mice were able to tolerate 25-100 millioncells per kg administration by intravenous infusion without any cardiacor pulmonary toxicity. LOT 11 cells were used for a tumorigenicity mousestudy. Mice were administered up to 250 million cells per kgsubcutaneously (SC) with no adverse effects.

6.2 Example 2: Improved Administrable Compositions

This Example demonstrates a formulation of human multipotent tissueculture plastic-adherent CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells,that even post-cryopreservation, represent homogenous, high viabilitycells suitable for administration to humans or animals. The cells of theformulation exhibit on average a 400-fold decrease in the amount ofaggregates formed, and an improved Maximum Tolerated Dose (MTD) ofapproximately 3-fold improvement over a previous formulation in anintravenous mouse model (data not shown). While the following exampledescribes formulation of isolated placental cells expressing particularsurface markers, the results presented herein indicate that the methodsand formulations can be used, and are compatible, with other cells,e.g., mammalian cells expressing different surface markers.

The formulation (designated “Formulation B”) comprises placental cellsof the above cellular phenotype at a concentration of 10±3×10⁶ cells/mL,in a solution containing 5.5% (w/v) Dextran 40, 10% (w/v) Human SerumAlbumin (HSA), and 5% (v/v) dimethyl sulfoxide (DMSO). The HSA andDextran 40 used herein are clinical grade; DMSO is GMP grade.

A Plasmalyte-based formulation designated “Formulation A,” is describedand compared to Formulation B, a Dextran 40 based formulation, in Table7. Cells are filtered in suspension through a 70 μm mesh in thepreparation of Formulation B, but are not filtered in preparation ofFormulation A.

TABLE 7 Composition of Formulation A and Formulation B Formulation AFormulation B Cell concentration 27 ± 8 × 10⁶ cells/mL 10 ± 3 × 10⁶cells/mL Bulk excipient Plasmalyte 5.5% Dextran 40 in saline DMSO 5%(v/v) 5% (v/v) concentration HSA concentration 10% 10%

Significant cell aggregation was observed when thawing Formulation A.Subsequent investigation showed that a number of parameters, includingthe composition of the formulation medium and the freezing concentrationof the cells were key contributors to cell aggregation. Optimizationstudies were conducted and Formulation B was designed specifically toreduce or eliminate these cell aggregation effects. In addition, thefiltration of the cell suspension through a 70 μm mesh as part of theprocess was introduced to provide better control of cell suspensionsduring formulation.

To quantify cell aggregates in the formulations, a filter retentionassay was devised as a development tool and used to compare a series ofplacental cell composition batches produced by both Formulation A andFormulation B. This became particularly critical since the cellaggregation in Formulation B was reduced to the point where it was nolonger reliably discernable by naked eye. A comparison of multiplesamples analyzed from representative batches of Formulation A andFormulation B is shown in Table 8. The method quantifies the pre-stainedcell aggregates retained on a filter from different samples by digitalimaging of the filter, and reports the area of filter (“Mean Area”)covered by the aggregates. As shown in Table 8, Formulation B hadconsistently less area coverage (aggregates) than Formulation A, withthe average difference being 400-fold. The data also show good processcontrol and reproducibility for Formulation B. Isolated placental cellcomposition lots of the two formulations used for in vivo studies arealso indicated in Table 16. The change from Formulation A to FormulationB improved the Maximum Tolerated Dose (MTD) approximately 3-fold in anintravenous in vivo mouse model (data not shown).

TABLE 8 Post-Thaw Aggregate formation in Formulation A and Formulation BFormulation Mean Area (in vivo testing Lot Bags Filters (px/MM) Std DevCV indicated by *) 1 2 13 97490 38369 0.39 Formulation A 2 1 12 9862933165 0.34 Formulation A* 1 1 2 63 81 1.28 Formulation B* 2 1 2 38 120.32 Formulation B 3 1 2 1539 573 0.37 Formulation B* 4 1 2 137 43 0.31Formulation B 5 3 6 125 162 1.29 Formulation B 6 3 6 71 57 0.81Formulation B Key: ″Bags″ indicates the number of isolated cellcomposition bags thawed and tested for that Lot; ″Filters″ indicates thetotal number of assay replicates for that Lot; ″Mean Area″ indicates themean area coverage of the filters for that Lot in units ofpixels/million cells applied to the filter; ″Std Dev″= standarddeviation; ″CV″= Coefficient of Variation.

6.3 Example 3: Characterization of Improved Administrable Compositions

This Example provides further characterization of pharmaceuticalformulations comprising HSA, dextran 40, and DMSO. Methods used in oneor more of the experiments described below are as follows:

Cellular viability assessment: Viability was assessed by a Trypan Blueexclusion assay using either a hemocytometer or a Vi-Cell cell viabilityanalyzer (Beckman Coulter, Fullerton, Calif.). Viability was expressedas a percentage viable cells out of total cells.

Cell counts: Cells were counted using either a hemocytometer or aVi-Cell cell viability analyzer (Beckman Coulter, Fullerton, Calif.).Cell counts are expressed as millions of cells per milliliter (MM/mL).

Cellular aggregation: The amount of cellular aggregation was measuredusing a Filter Retention Assay (FRA), which measures the amount ofcellular aggregation by staining cells and passing them through a 70 μmfilter. Cellular aggregates greater than 70 μm cannot pass the filterand are quantified by image analysis using a Vi-Cell cell viabilityanalyzer (Beckman Coulter, Fullerton, Calif.). Data are expressed inpixels per million cells loaded (px/MM).

Flow cytometry: Cells were assessed for the levels of the cellularmarkers CD10, CD34, CD105 and CD200 by flow cytometry. Values areexpressed as a percentage of cells positive and/or negative for aparticular marker, or combination of markers.

Immunosuppression: The immunosuppressive activity of cells in theformulations described below was assessed using a Bead T-cell Reaction(BTR) assay, which measures the ability of cells to suppress a T-cellresponse to antigenic beads.

While the following example describes formulation of isolated placentalcells expressing particular surface markers, the results presentedherein indicate that the methods and formulations can be used, and arecompatible, with other cells, e.g., mammalian cells expressing differentsurface markers.

6.3.1 Characterization of DMSO and Dextran:HSA Ratios

This example describes the effect of varying the concentrations of HSA,Dextran, and DMSO, and of varying the ratio of dextran to HSA, oncellular aggregation, cell viability and recovery, and cell phenotypeand functionality.

Experimental Conditions

The three component (HSA, dextran 40 and DMSO) solution space for thecell formulation was investigated at the conditions shown on the ternarydiagram in FIG. 1. Experimental conditions were chosen along two axes:one which tested different percentages of DMSO while holding theDextran:HSA ratio constant (see formulations 1-4, below), and anotherthat varied the ratio of Dextran:HSA while holding the percentage ofDMSO constant (see formulations 3, 5-8, below).

Methods and Materials

Approximately twelve million cryopreserved CD10⁺, CD34⁻, CD105⁺, CD200⁺placental multipotent cells were expanded in Nunc™ 10-tray CellFactories, one for each of the formulations below, to approximately1.2×10⁸ cells. Cells were harvested by incubation for 10 min. at roomtemperature with 0.25% Trypsin/EDTA. Dissociated cells were transferredto a 500 mL centrifuge tube containing 250 mL of 2% fetal bovine serum(FBS) in Dulbecco's Modified Eagle's Medium (DMEM). Cells were thencentrifuged at 1040 RPM in 500 mL tubes in a Sorvall RC3BP centrifuge.Cells were re-suspended in a 0.9% saline/5% dextran 40 solution. Thecells were then centrifuged at 1420 RPM for 10 min, and suspended in 10mL of one of the following eight formulations (dextran:HSA ratios aredisplayed in units of “volume fraction of 25% HSA” (VF HSA) forformulations and 5-8):

Formulation 1: 20% DMSO, 8.5% HSA, 4.6% dextran 40Formulation 2: 10% DMSO, 9.5% HSA, 5.2% dextran 40Formulation 3: 5% DMSO, 10% HSA, 5.5% dextran 40 (VF HSA=0.4) (controlformulation)Formulation 4: 0% DMSO, 10.5% HSA, 5.8% dextran 40Formulation 5: 5% DMSO, 0% HSA, 9.5% dextran 40 (VF HSA=0)Formulation 6: 5% DMSO, 3.125% HSA, 8.25% dextran 40 (VF HSA=0.125)Formulation 7: 5% DMSO, 16.88% HSA, 2.75% dextran 40 (VF HSA=0.675)Formulation 8: 5% DMSO, 23.75% HSA, 0% dextran 40 (VF HSA=0.95)

The formulations were prepared from the following stock solutions: 100%DMSO (Bioniche Pharma, Belleville, Ontario), 25% HSA (Octapharma,Hoboken, N.J.) and 10% dextran 40 in 0.9% Saline (Hospira, Lake Forest,Ill.).

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ cells/filter. Cells were cryopreserved in a Thermo control ratefreezer to −70° C. at a concentration of 7.5×10⁶ cells/mL. Cells werethawed prior to use, and samples were processed shortly after thaw. A100 μL sample of the undiluted cell suspension was taken post thaw,diluted with 900 μL of phosphate buffered saline (PBS), and cell countswere performed in duplicate using a Vi-Cell cell viability analyzer(Beckman Coulter, Fullerton, Calif.) and reported as numbers of pixelsper million cells (higher numbers of pixels indicate higher numbers ofcellular aggregates).

Cell viability assay (MTS assay): Cell viability was determined using aCellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega,Madison, Wis.). Cells in 96-well plates were combined with(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(MTS) and phenazine methosulfate (PMS), an electron coupling reagent,according to manufacturer's directions. Absorbance at 490 nm of theresulting formazan product, produced by cellular bioreduction of MTS,was then determined.

Annexin well plate assay: Thawed cells were diluted to a concentrationof 1×10⁵ cells/mL in an Annexin-V Labeling solution (Roche, Cat. No. 11828 681 001). 100 μL of the cell suspension was added to a 96 well platein triplicate and incubated for 15 minutes at room temperature with noexposure to light. After 15 minutes, three different positions withineach well were imaged under brightfield and fluorescent light(excitation wavelength of 488 nm and detection at wavelength 528 nm). Anautomated cell counting software (Axiovision) was used to count totalcells per image. Apoptotic/necrotic cell populations were determined bycounting Annexin positive cells at each position (fluorescent image) anddividing by total cells at each position (brightfield image).Apoptotic/necrotic cell populations per condition were determined byaveraging the computed populations in three different well positions of3 wells.

Results Cellular Aggregation

Across varying percentages of DMSO, e.g., 0 to 20 percent (formulations1-4), no effect on cellular aggregation was observed, as shown in FIG.2. All DMSO conditions exhibited levels of aggregation within the rangeobserved for the control formulation comprising 5% DMSO, 5.5% dextran 40and 10% HSA. HSA and Dextran concentrations were also varied at aconstant 5% DMSO concentration (FIG. 3), with conditions ranging from noHSA (HSA volume fraction=0) to no Dextran (HSA volume fraction=0.95).FRA values across volume fractions of 0.125 to 0.95 of 25% HSA (finalconcentration 3.125% to 23.75% HSA, respectively) indicated minimalaggregation. Observed levels of aggregation in the presence of HSA wereequal to or below the control formulation comprising 5% DMSO, 5.5%dextran 40 and 10% HSA.

Post Thaw Viability and Recovery

Post thaw viability (FIG. 4) and post thaw recovery (FIG. 5) acrossdifferent percentages of DMSO (0, 5, 10 and 20%) was measured throughtrypan blue exclusion, using the Vi-Cell automated cell counter, toassess the cryoprotective capabilities of each formulation. Post thawviability was greater than 90% with formulations comprising 0%, 5% and10% DMSO, respectively, with a maximum value observed at 5% DMSO, whileviability was below 75% with a formulation comprising 20% DMSO. Culturere-establishment was measured through the MTS assay. Culturere-establishment was observed to be maximal at 5% DMSO (FIG. 6).

The viability profile across the different volume fractions of 25% HSA,i.e., differing ratios of dextran:HSA, is shown in FIGS. 7-9. Post thawviability, as determined by Trypan blue exclusion, exhibited valuesranging from 83.5% to 98.5% across the various volume fractions of HSA(FIG. 7). A maximum value was achieved at a 0.40 fraction of 25% HSA,i.e., 10% HSA: 5.5% Dextran 40. Post thaw cell recovery was alsocalculated, and values ranged from 80% to 120%, though samplescomprising at least 0.2 volume fraction of 25% HSA exhibited valuesranging from 100% to 120% (FIG. 8). The culture re-establishment data,as determined by MTS assay, exhibit a similar profile to that of postthaw viability, with maximum value occurring at a 0.125 fraction of 25%HSA, i.e., 3.13% HSA: 8.25% Dextran 40 (FIG. 9).

Trypan viability does not have the ability to detect apoptotic cells.However analysis of the cell size distributions may indicate changes incell health. To estimate the percent of apoptotic cells in cellpopulations thawed from cells frozen in the absence of DMSO, a post thawwell plate annexin assay was conducted. The assay estimated that 51% ofthe cells frozen in 0% DMSO were apoptotic as opposed to 15% apoptoticwhen frozen in 5% DMSO (data not shown).

Phenotype and Functionality

The ability to maintain the cells' CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype was tested across the different percentages of components byflow cytometry. Cells formulated in formulation 2 (10% DMSO, 9.5% HSA,5.2% dextran 40), formulation 5 (5% DMSO, 0% HSA, 9.5% dextran 40),formulation 6 (5% DMSO, 3.13% HSA, 8.25% dextran 40) and formulation 7(5% DMSO, 16.88% HSA, 2.75% dextran 40) maintained this phenotypeapproximately as well as control formulation 3, and had CD105⁺/CD200⁺values between 80.3% and 84.5%. Additionally, CD10⁺/CD34⁻ expressionwas >95% for each formulation. Conditions 1, 4 and 8 indicated a changein the cells' physical characteristics, possibly because debris affectedthe flow cytometric assay.

Cell functionality was assessed through the Bead T cell Reaction (BTR)assay, which measures the ability of cells to suppress a T cell responseto antigenic beads (FIG. 10). Formulation 6 (5% DMSO, 3.13% HSA, 8.25%dextran 40), and formulation 7 (5% DMSO, 16.88% HSA, 2.75% dextran 40)had suppression within 1 standard deviation of a control formulation 3comprising 5% DMSO/10% HSA and 5.5% dextran 40. These three samples hadthe highest trypan blue viabilities and culture re-establishment values.The other samples had lower levels of suppression, with a reduction thatgenerally correlated with decreased MTS.

Conclusions:

Formulations comprising DMSO concentrations ranging from 0 to 20%exhibit levels of cellular aggregation comparable to that observed for acontrol formulation comprising 5% DMSO, 10% HSA, and 5.5% dextran 40.However, cell viability was reduced when cells were cryopreserved informulation comprising 20% DMSO, and cells frozen in 0% DMSO exhibitedsignificantly enhanced apoptosis post-thaw relative to cells frozen in5% DMSO. There was no appreciable degradation of CD10⁺, CD34⁻, CD105⁺,CD200⁺ phenotype for formulations comprising 5% and 10% DMSO. Thus, theresults above demonstrate that use of formulations comprising 5-10% DMSOis preferred for maintaining post-thaw viability.

With respect to varying the ratio of HSA:dextran, formulationscomprising 23.75% HSA, 0% dextran exhibited some reduction in post-thawviability and culture re-establishment, while post-thaw viability,immunosuppressive activity and re-establishment values were highest forformulations comprising dextran:HSA ratios of: (i) 3.13% HSA to 8.25%dextran; (ii) 10% HSA to 5.5% dextran; and (iii) 16.88% HSA to 2.75%dextran. Thus, these results demonstrate that the HSA:dextran ratio ofthe formulations described herein may be varied within a defined a rangewithout an appreciable loss of cell viability, cell recovery afterthawing, degradation of CD10⁺, CD34⁻, CD105⁺, CD200⁺ phenotype, orimmunosuppressive activity, relative to formulations comprising anHSA:dextran ratio of 10% HSA and 5.5% Dextran 40. The data presentedherein support a working range of HSA:dextran ratios of at least betweenabout 6:1 HSA:dextran to about 1:2.6 HSA:dextran.

6.3.2 Effect of Freezing Cell Density

This example describes the effects of cell concentration, e.g., freezingcell densities ranging from 1−40×10⁶ cells/mL, on cell viability; CD10⁺,CD34⁻, CD105⁺, CD200⁺ phenotype; cellular aggregation; andimmunosuppressive functionality of the cells.

Methods and Materials

CD10⁺, CD34⁻, CD105⁺, CD200⁺ placental stem cells were cultured for 3days, harvested, centrifuged, and re-suspended in formulation comprising5% DMSO, 5.5% dextran 40, 10% HSA to a concentration of about 3.5×10⁷cells/mL, and filtered through a 70 μm filter. The post-filter cellswere serially diluted in the above formulation to create cell samplescomprising, in sterile bags, the following cell densities: 1×10⁶cells/mL, 7.5×10⁶ cells/mL, 15×10⁶ cells/mL, and 20×10⁶ cells/mL. Cellswere harvested separately to create a separate cell sample comprising4.0×10⁷ cells/mL. Cells of the 4.0×10⁷ cells/mL sample were re-suspendedin 5 mL of formulation comprising 5% DMSO, 5.5% dextran 40, 10% HSA to aconcentration of about 4.6×10⁷ cells/mL, filtered through a 70 μmfilter, and diluted to 4.0×10⁷ cells/mL in a 20 mL bag using the aboveformulation. One 20 mL bag of the 4.0×10⁷ cells/mL sample, duplicate 20mL bags of the 1×10⁶ cells/mL, 7.5×10⁶ cells/mL, 15×10⁶ cells/mL, and20×10⁶ cells/mL samples, and five 280 μL vials of each sample werefrozen at −70° C. using a controlled rate freezer. These samples werelater thawed and analyzed to determine the effects of freezingconcentration on various cellular characteristics, including (1) cellcount; (2) viability; (3) phenotype; (4) cell aggregation; and (5)potency.

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ cells/filter. Cells were cryopreserved in a Thermo control ratefreezer to −70° C. at a concentration of 7.5×10⁶ cells/mL. Cells werethawed prior to use, and samples were processed shortly after thaw. A100 μL sample of the undiluted cell suspension was taken post thaw,diluted with 900 μL of phosphate buffered saline (PBS), and cell countswere performed in duplicate using a Vi-Cell cell viability analyzer(Beckman Coulter, Fullerton, Calif.) and reported as numbers of pixelsper million cells (higher numbers of pixels indicate higher numbers ofcellular aggregates).

Results: Cell Count/Viability

One to two 1 mL samples from each thawed bag were taken for viable cellcounts and viability determination using a Vi-Cell cell viabilityanalyzer (Beckman Coulter, Fullerton, Calif.) according tomanufacturer's directions. The average viability remained constant forall the conditions, at about 97%. The viable cell count corresponded tothe initial freezing concentration (see Table 9 below).

TABLE 9 Vi-Cell viable cell concentration and viability Freezing Cellcount (× 10⁶) Viability Concentration cells/mL (%)  1 × 10⁶/mL 1.2098.70 7.5 × 10⁶/mL  8.32 97.12 15 × 10⁶/mL 16.47 97.33 20 × 10⁶/mL 20.8097.61 40 × 10⁶/mL 39.92 96.71

Phenotype

The ability to maintain the cells' CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype was tested across the different cell densities by flowcytometry. As presented in Table 10, the phenotype does not changebetween different freezing concentrations. CD200⁺/CD105⁺ expressionremained around 86% and CD34⁻/CD10⁺ expression remained around 99% forall conditions.

TABLE 10 CD200+/CD105+ and CD34−/CD10+ expression Freezing concentrationCD200⁺/CD105⁺ CD34⁻/CD10⁺  1 × 10⁶/mL 87.3 98.2 7.5 × 10⁶/mL  86.7 99.115 × 10⁶/mL 85.8 99.1 20 × 10⁶/mL 85.5 98.9 40 × 10⁶/mL 86.3 98.5Negative control 69.4 98.5 Positive control 91.1 98.8

Cellular Aggregation

Replicates of each condition were analyzed by a Filter Retention Assayto determine the degree of cellular aggregation at different freezingconcentrations (FIG. 11). Duplicates of the assay were performed for the1×10⁶ cells/mL, 7.5×10⁶ cells/mL, 15×10⁶ cells/mL and 20×10⁶/mL samples.One cell sample was assayed in duplicate for the 40×10⁶ cells/mL sample.The 40×10⁶ cells/mL sample produced the highest cellular aggregationsignal for all cell densities tested. All of the other samples were ator below the amount of aggregation observed with a control samplepreviously cryopreserved at 7.5×10⁶ cells/mL in 5% DMSO, 5.5% dextran40, 10% HSA.

An additional, separate cellular aggregation assay was performed to testcells cryopreserved in formulation comprising 5% DMSO, 5.5% dextran 40,10% HSA at 7.5×10⁶/mL and 20×10⁶/mL (FIG. 12). The additional datashowed an increased signal at 20×10⁶/mL, indicating that cells frozen at20×10⁶/mL give variable cellular aggregation results. As a result, cellsfrozen at this concentration or higher have an increased potential foraggregation. These data demonstrate that aggregation increases withincreasing cell concentration, and a freezing cell density of 20×10⁶ mLcan show increases in cellular aggregation relative to a freezing celldensity of 7.5×10⁶/mL.

Functionality

A vial from each condition was used for mixed leukocyte reaction (MLR)and bead T-cell reaction (BTR) analysis to assess the immunomodulatoryproperties of the cells at varying freezing cell densities, as measuredby suppression of proliferation of CD4⁺ T cells and CD8⁺ T cells. TheMLR results indicate a dip in CD4 and CD8 suppression at 15×10⁶ cells/mLand 40×10⁶ cells/mL, though suppression at 20×10⁶ cells/mL wascomparable to that observed at 1×10⁶ cells/mL and 7.5×10⁶ cells/mL.However, the BTR results show a reduction in CD4 and CD8 suppression at20×10⁶ cells/mL and 40×10⁶ cells/mL.

TABLE 11 MLR and BTR results - percent T cell reactivity compared to Tcell reactivity in the absence of placental stem cells. MLR BTR CD4 CD8CD4 CD8 Sample Suppression Suppression Suppression Suppression  1 ×10⁶/mL 63 64 63 62 7.5 × 10⁶/mL  63 64 63 62 15 × 10⁶/mL 49 46 61 64 20× 10⁶/mL 65 65 36 39 40 × 10⁶/mL 40 45 33 38

Conclusions:

The results above demonstrate that cell viability and CD10⁺, CD34⁻,CD105⁺, CD200⁺ phenotype does not change as a function of freezing celldensity. However, cells cryopreserved at a density of 20×10⁶ cells/mLdemonstrated a variable increase in cellular aggregation and a variabledecrease in immunosuppressive activity, while cells cryopreserved at adensity of 40×10⁶ cells/mL exhibited a consistent increase in cellularaggregation and a consistent decrease in immunosuppressive activity. Assuch, while cells may be formulated in the formulations described hereinat a density of up to, e.g., 40×10⁶ cells/mL without an appreciabledecrease in cell viability or in the number of cells displaying a CD10⁺,CD34⁻, CD105⁺, CD200⁺ phenotype, a freezing cell density in the range of1.0-15×10⁶ cells/mL is preferable for minimizing cellular aggregationupon thaw.

6.3.3 Effect of Molecular Weight of Dextran

This Example describes the effect of varying molecular weights ofdextran, e.g., dextran 1 (MW=1000), dextran 40 (MW=40,000) and dextran70 (MW=70,000) on cellular aggregation, viability, recovery, CD10⁺,CD34⁻, CD105⁺, CD200⁺ phenotype and functionality.

Materials and Methods

Twelve million cryopreserved CD10⁺, CD34⁻, CD105⁺, CD200⁺ placentalmultipotent cells were expanded in Nunc™ 10-tray Cell Factories, one foreach of the formulations below, to approximately 1.2×10⁸ cells. Cellswere harvested by incubation for 10 min. at room temperature with 0.25%Trypsin/EDTA. Dissociated cells were transferred to a 500 mL centrifugetube containing 250 mL of 2% fetal bovine serum (FBS) in Dulbecco'sModified Eagle's Medium (DMEM). Cells were then centrifuged at 1040 RPMin 500 mL tubes in a Sorvall RC3BP centrifuge. Cells were re-suspendedin a 0.9% saline/5% dextran 40 solution. The cells were then centrifugedat 1420 RPM for 10 min, and suspended in 10 mL of the followingformulations:

Formulation 1: 5% DMSO, 5.5% dextran 40 (Hospira, Lake Forest, Ill.),10% HSA (control)

Formulation 2: 5% DMSO, 5.5% Dextran 1 (Pharmacosmos), 10% HSA

Formulation 3: 5% DMSO, 5.5% dextran 40 (Pharmacosmos), 10% HSA

Formulation 4: 5% DMSO, 5.5% Dextran 70 (Pharmacosmos), 10% HSA

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ cells/filter. Cells were cryopreserved at a concentration of7.5×10⁶ cells/ml in a Thermo control rate freezer to −70° C. Cells werethawed prior to use, and samples were processed shortly after thaw. A100 μL sample of the undiluted cell suspension was taken post thaw,diluted with 900 μL of phosphate buffered saline (PBS), and cell countswere performed in duplicate using a Vi-Cell cell viability analyzer(Beckman Coulter, Fullerton, Calif.).

Results: Cellular Aggregation

Cellular aggregation within each formulation was assessed using theFilter Retention Assay. Formulation 2 (dextran 1), formulation 3(dextran 40) and formulation 4 (dextran 70) demonstrated cellaggregation rates equivalent to, or below, control formulation 5% DMSO,5.5% dextran 40, and 10% HSA (FIG. 13).

Viability and Recovery

The post thaw viability for samples comprising dextran 1, dextran 40 ordextran 70 ranged from 96.0% to 97.7% viable (FIG. 14). Similarly, postthaw recovery was comparable to that of the control formulation, withcell viabilities ranging from 92% to 109% of the control formulation(FIG. 15).

Phenotype and Functionality

The ability to maintain the cells' CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype was tested across the different dextran molecular weights byflow cytometry, and cell immunosuppressive capability was tested in aBTR assay. CD200⁺/CD105⁺ expression across all formulations testedranged from 89.1% to 91.6%, and CD34⁻/CD10⁺ expression was >95% for allconditions (FIG. 16). Furthermore, CD4⁺ and CD8⁺ T-cell suppressionacross the different dextran molecular weights fell within 1 standarddeviation of an expected value derived from 4 different experimentalreplicates of a standard control formulation comprising dextran 40 (FIG.17).

Conclusions:

These results demonstrate that dextran 1 or dextran 70 may besubstituted for dextran 40 in the formulations described herein, withoutimpacting cellular aggregation, viability, recovery, phenotype orimmunosuppressive capability.

6.3.4 Effect of Different Polysaccharides

The Example describes the effect of polysaccharides other than dextran40 in the cell formulation on cell viability and proliferation.

Materials and Methods

Twelve million cryopreserved CD10⁺, CD34⁻, CD105⁺, CD200⁺ placentalmultipotent cells were expanded in Nunc™ 10-tray Cell Factories, one foreach of the formulations below, to approximately 1.2×10⁸ cells. Cellswere harvested by incubation for 10 min. at room temperature with 0.25%Trypsin/EDTA. Dissociated cells were transferred to a 500 mL centrifugetube containing 250 mL of 2% fetal bovine serum (FBS) in Dulbecco'sModified Eagle's Medium (DMEM). Cells were then centrifuged at 1040 RPMin 500 mL tubes in a Sorvall RC3BP centrifuge. Cells were re-suspendedin a 0.9% saline/5% dextran 40 solution. The cells were then centrifugedat 1420 RPM for 10 min, and suspended in 10 mL of the followingformulations:

Formulation 1: 5% DMSO, 5.5% dextran 40, 10% HSA (Control)

Formulation 2: 5% DMSO, 5.5% Maltodextrin, 10% HSA Formulation 3: 5%DMSO, 5.5% Sucrose, 10% HSA Formulation 4: 5% DMSO, 5.5% Trehalose, 10%HSA Formulation 5: 5% DMSO, 55USP/mL Heparin, 10% HSA Formulation 6: 5%DMSO, 3.3% Hetastarch, 10% HSA Formulation 7: 5% DMSO, 5.5% Glycogen,10% HSA

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ cells/filter. Cells were cryopreserved in a Thermo control ratefreezer to −70° C. Cells were thawed prior to use, and samples wereprocessed shortly after thaw. A 100 μL sample of the undiluted cellsuspension was taken post thaw, diluted with 900 μL of phosphatebuffered saline (PBS), and cell counts were performed in duplicate usinga Vi-Cell cell viability analyzer (Beckman Coulter, Fullerton, Calif.).

Results: Cellular Aggregation

Cellular aggregation within each formulation was assessed using theFilter Retention Assay. Formulations 2-7 were determined to produce cellaggregation equivalent to, or below, control formulation 5% DMSO, 5.5%dextran 40, and 10% HSA (FIG. 18). Formulation 4, comprising trehalose,formulation 5, comprising heparin, and formulation 7, comprisingglycogen, demonstrated cell aggregation rates substantially below thecontrol formulation.

Post Thaw Viability and Recovery

Post thaw viability, viable cell recovery and cell size data wasassessed across each of formulations 1-7 to assess the formulations'cryoprotective capabilities. Post thaw viability was assessed by trypanblue exclusion Vi-Cell cell viability analyzer (Beckman Coulter,Fullerton, Calif.). Post thaw viability ranged from 95.2% to 98.6%, withsucrose and glycogen on the lower end of the range (FIG. 19). Thecontrol dextran formulation resulted in cell 98% viability. Post thawviable cell recovery was calculated to understand cell losses across thefreeze/thaw process, values ranged from 84% to 115% across the differentpolysaccharides (FIG. 20).

Phenotype and Functionality

The ability to maintain the cells' CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype was tested across the different polysaccharides by flowcytometry. Cells formulated in formulations 2-4 and 6 maintained thisphenotype approximately as well as control formulation 1, and were 89.4%to 92.9% CD105⁺/CD200⁺. CD10⁺/CD34⁻ expression was >95% for eachformulation. Of cells frozen in heparin, 85.4% maintained theCD105⁺/CD200⁺ phenotype (FIG. 21).

Cell functionality was assessed through the Bead T cell Reaction (BTR)assay, which measures the ability of cells to suppress a T cell responseto antigenic beads. Cells formulated in sucrose exhibited decreasedT-cell suppression compared to that of the within 1 standard deviationof an expected value derived from 4 different experimental replicates ofa standard control formulation comprising dextran 40 (FIG. 22). Acrossthe other polysaccharides, CD4 and CD8 T-cell suppression fell within 1standard deviation of the expected value.

Conclusions:

With respect to cell aggregation, post-thaw viability, post-thaw cellrecovery and maintenance of phenotype, use of formulations comprisingmaltodextran, trehalose and hetastarch resulted in placental cellpopulations having approximately the same characteristics as use of thedextran 40 formulation. As such, while formulations comprising sucrose,heparin or glycogen may be used to formulate CD10⁺, CD34⁻, CD105⁺,CD200⁺ placental stem cells, the use of formulations comprising dextran40, maltodextran, trehalose or hetastarch is preferred.

6.3.5 Effect of Protein Alternatives of HSA

This Example demonstrates that in a 5.5% dextran 40, 10% HSA and 5% DMSOformulation, the human serum albumin concentration may be reduced, andthat the HSA may be substituted with bovine serum albumin or fetalbovine serum.

Formulation 1 (F1) is comprised of 5.5% dextran 40, 10% HSA and 5% DMSO.In order to understand the role of HSA within F1 and understand itsimpact on cell compositions, alternative formulations with similarproteins similar to human serum albumin, i.e., bovine serum albumin(BSA) and fetal bovine serum (FBS), were tested.

Materials and Methods

Twelve million cryopreserved CD10⁺, CD34⁻, CD105⁺, CD200⁺ placentalmultipotent cells were expanded in Nunc™ 10-tray Cell Factories, one foreach of the formulations below, to approximately 1.2×10⁸ cells. Cellswere harvested by incubation for 10 minutes at room temperature with0.25% Trypsin/EDTA. Dissociated cells were transferred to a 500 mLcentrifuge tube containing 250 mL of 2% fetal bovine serum (FBS) inDulbecco's Modified Eagle's Medium (DMEM). Cells were then centrifugedat 1040 RPM in 500 mL tubes in a Sorvall RC3BP centrifuge. Cells werere-suspended in a 0.9% saline/5% dextran 40 solution. The cells werethen centrifuged at 1420 RPM for 10 min, and suspended in 10 mL of thefollowing formulations:

-   -   Formulation 1: 5% DMSO, 5.5% dextran 40, 10% HSA (control)    -   Formulation 2: 5% DMSO, 5.5% dextran 40, 4% HSA    -   Formulation 3: 5% DMSO, 5.5% dextran 40, 10% BSA    -   Formulation 4: 5% DMSO, 5.5% dextran 40, 10% FBS

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ million cells/filter. Cells were cryopreserved in a Thermocontrol rate freezer to −70° C. Cells were thawed prior to use, andsamples were processed shortly after thaw. A 100 JAL sample of theundiluted cell suspension was taken post thaw, diluted with 900 μL ofphosphate buffered saline (PBS), and cell counts were performed induplicate using a Vi-Cell cell viability analyzer (Beckman Coulter,Fullerton, Calif.).

Results: Cellular Aggregation

Cellular aggregation, as measured by the Filter Retention Assay (FRA),was at or under 100 pixels per square millimeter for all conditions(FIG. 23). However, 10% HSA and 10% BSA appeared to produce discernablyfewer aggregates than 4% HSA and 10% FBS.

In order to understand the cryoprotective capability of each solution,post thaw viability, recovery and cell size was assessed through the useof Trypan Blue exclusion on a Vi-Cell cell viability analyzer (BeckmanCoulter, Fullerton, Calif.). Post thaw viability across each conditionwas within assay variability and ranged from 95% to 98% viable (FIG.24). Similarly, post thaw cell recovery was comparable to that of the10% HSA control, with values ranging from 100 to 127% (FIG. 25).

Phenotype and Functionality

The ability to maintain the cells' CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype was tested across the different formulations by flowcytometry. (FIGS. 26 and 27). Cells formulated in formulations 2-4maintained this phenotype approximately as well as the control 10% HSAformulation, with values ranging from 88.2% to 93.5% of tested cells.CD10⁺/CD34⁻ expression was >95% for each formulation.

Cell functionality was measured through the Bead T-cell Reaction assay.Compared to the other conditions suppression of CD4 and CD8 T-cells waselevated when cells were formulated in FBS with values falling within1.5 standard deviations of the expected value derived from 4 differentexperimental replicates of a standard control formulation comprising 10%HSA (FIG. 28). Across all other conditions suppression was within 1standard deviation of the expected value.

Conclusions:

The results above demonstrate that 4% HSA, 10% BSA, or 10% FBS may besubstituted for 10% HSA without appreciable loss of cell viability, cellrecovery after thawing, degradation of CD10⁺, CD34⁻, CD105⁺, CD200⁺phenotype, or immunosuppressive activity. Thus, a range of at leastbetween about 4% to about 10%, HSA, BSA and/or FBS is particularlysuitable for the formulations described herein.

6.3.6 Compatibility with Different Cell Types

The Example demonstrates that bone marrow-derived mesenchymal stem cellsand natural killer cells can be formulated in the same manner asplacental multipotent cells. Thus, this Example shows that other cells,in addition to placental multipotent cells, can be formulated in themanner presented herein.

Methods and Materials:

Bone marrow-derived mesenchymal stem cells (BMMSCs) and natural killer(NK) cells were expanded and harvested using the following formulations:

-   -   F1: Culture of cells for 3 days, followed by collection and        resuspension of cells in 5% DMSO, 5.5% dextran 40, 10% HSA,        filtration of cells through a 70 μm filter, and cryopreservation        of cells at approximately 7.5×10⁶ cells/mL; and    -   F2: Culture of cells for four days, followed by collection and        resuspension of cells in Plasmalyte A comprising 5% DMSO and 10%        HSA.

Cells were frozen at −70° C. using a controlled rate freezer. BMMSCswere cryopreserved in 20 mL bags in F2 formulation and 10 mL bags in F1formulation; and CD3⁻, CD56⁺ NK cells were cryopreserved in 10 mL bagsin F2 formulation and in 1.5 mL vials in F1 formulation. These sampleswere later thawed and analyzed to determine the effects of freezingformulation on cellular characteristics.

Filter Retention Assay: Cellular aggregation was assessed through theuse of filter retention assay. Cell concentration was adjusted toapproximately 1.2×10⁷ cells/mL prior to filtration. Filter load (numberof cells per unit filter area) was held constant at approximately2.4×10⁸ million cells/filter. Cells were cryopreserved in a Thermocontrol rate freezer to −70° C. Cells were thawed prior to use, andsamples were processed shortly after thaw. A 100 μL sample of theundiluted cell suspension was taken post thaw, diluted with 900 μL ofphosphate buffered saline (PBS), and cell counts were performed induplicate using a Vi-Cell cell viability analyzer (Beckman Coulter,Fullerton, Calif.).

Results Viability

Post-thaw samples were used to determine the viability of the cellsfrozen under different conditions. Viability of BMMSCs and NK cells wasnot significantly affected by the freezing conditions.

Phenotype

NK markers were assayed to determine the effects of F1 and F2formulations on the NK phenotype (CD3⁻, CD56⁺). The two formulationsused did not significantly affect the percentage of NK cells displayingthe NK phenotype. BMMSC were also analyzed for the expression of CD10,CD34⁻, CD44, CD45, CD90, CD98, CD105, CD117, CD166, CD200,Pan-cytokeratin, and KDR. The expression, or lack of expression, ofthese markers did not vary significantly in BMMSC formulated in F1 or F2formulations.

Cellular Aggregation

In the Filter Retention Assay, two replicates of each of the BMMSCformulations, and one assay replicate of the NK formulations, wereperformed. The F1 formulation produced significantly fewer aggregatesthan the F2 formulation for both BMMSCs and NK cells; the effect offormulation F2, however, was more pronounced for BMMSCs than for NKcells (FIG. 29).

Conclusions:

The results above demonstrate that bone marrow-derived mesenchymal stemcells and natural killer cells can successfully be formulated in 5%DMSO, 5.5% dextran 40, 10% HSA, with cellular aggregation, viability andphenotype retention similar to that of CD10⁺, CD34⁻, CD105⁺, CD200⁺placental multipotent cells in the same formulation. In addition, forthe placental multipotent cells, Plasmalyte-containing formulations ofBMMSCs and NK cells show significantly higher rates of cellularaggregation; as such, dextran-containing formulations are preferred.

EQUIVALENTS

The compositions and methods disclosed herein are not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the compositions and methods in addition to thosedescribed will become apparent to those skilled in the art from theforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1-28. (canceled)
 29. A composition comprising a plurality of isolatedhuman adherent placental cells in a solution comprising 10% dextran 40,wherein said composition comprises between about 1.0±0.3×106 cells permilliliter to about 5.0±1.5×106 cells per milliliter, and wherein saidcomposition comprises no macro cell clumps.
 30. The composition of claim29, wherein said placental stem cells are thawed cryopreserved cells.31. The composition of claim 29, wherein said composition is containedwithin a bag.
 32. The composition of claim 29, wherein said compositioncomprises no macro cell clumps.
 33. The composition of claim 29, whereinsaid composition comprises fewer than about 200 micro cell clumps per106 cells.
 34. The composition of claim 29, wherein said compositioncomprises fewer than about 150 micro cell clumps per 106 cells.
 35. Thecomposition of claim 29, wherein said composition comprises fewer thanabout 100 micro cell clumps per 106 cells.
 36. The composition of claim29, wherein said isolated human adherent placental cells are CD10+,CD34− and CD105+.
 37. The composition of claim 36, wherein said CD10+,CD34− and CD105+ cells are CD200+.
 38. The composition of claim 37,wherein said CD10+, CD34−, and CD105+ and CD200+ cells are either CD45−or CD90+.
 39. The composition of claim 37, wherein said CD10+, CD34−,and CD105+ and CD200+ cells are CD45− or CD90+.
 40. The composition ofclaim 29, wherein said composition is a pharmaceutical composition.41-42. (canceled)